Targeted nanoparticles

ABSTRACT

Described herein are carrier nanoparticles comprising a polymer containing a polyol coupled to a polymer containing a boronic acid, configured to present the polymer containing a boronic acid to an environment external to the nanoparticle. Targeted versions of the described nanoparticles are also described, as are related compositions, methods and systems.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No. CA151819and under Grant No. CA119347, awarded by the National Institutes ofHealth. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 23, 2013, isnamed Sequence_Listing_CRF_CTEK0113 and is 1,485 bytes in size.

TECHNICAL FIELD

The present disclosure relates to carrier nanoparticles and inparticular to nanoparticles suitable for delivering compounds ofinterest, and related compositions, methods and systems.

BACKGROUND

Effective delivery of compounds of interest to cells, tissues, organs,and organisms has been a challenge in biomedicine, imaging and otherfields where delivery of molecules of various sizes and dimensions to apredetermined target is desirable.

Whether for pathological examination, therapeutic treatment or forfundamental biology studies, several methods are known and used fordelivering various classes of biomaterials and biomolecules which aretypically associated with a biological and/or chemical activity ofinterest.

As the number of molecules suitable to be used as chemical or biologicalagents (e.g. drugs, biologics, therapeutic or imaging agents) increases,development of a delivery systems suitable to be used with compounds ofvarious complexity, dimensions and chemical nature has proven to beparticularly challenging.

Nanoparticles are structures useful as carriers for delivering agentswith various methods of delivery. Several nanoparticle delivery systemsexist, which utilize an array of different strategies to package,transport, and deliver an agent to specific targets.

SUMMARY

Provided herein are nanoparticles and related compositions, methods andsystems that in several embodiments provide a multifunctional tool foreffective and specific delivery of a compound of interest. Inparticular, in several embodiments, nanoparticles herein described canbe used as a flexible system for carrying and delivering a wide range ofmolecules of various sizes, dimensions and chemical nature topredetermined targets.

According to one aspect, a nanoparticle comprising a polymer containinga polyol and to a polymer containing a boronic acid is described. In thenanoparticle, the polymer containing a boronic acid is coupled to thepolymer containing a polyol and the nanoparticle is configured topresent the polymer containing a boronic acid to an environment externalto the nanoparticle. One or more compounds of interest can be carried bythe nanoparticle, as a part of or attached to the polymer containing apolyol and/or the polymer containing a boronic acid.

According to another aspect, a composition is described. The compositioncomprises a nanoparticle herein described and a suitable vehicle and/orexcipient.

According to another aspect, a method to deliver a compound to a targetis described. The method comprises contacting the target with ananoparticle herein described wherein the compound is comprised in thepolymer containing a polyol or in the polymer containing a boronic acidof the nanoparticle herein described.

According to another aspect, a system to deliver a compound to a targetis described. The system comprises at least a polymer containing apolyol and polymer containing a boronic acid capable of reciprocalbinding through a reversible covalent linkage, to be assembled in ananoparticle herein described comprising the compound.

According to another aspect, a method to administer a compound to anindividual is described. The method comprises administering to theindividual an effective amount of a nanoparticle herein described,wherein the compound is comprised in the polymer containing a polyoland/or in the polymer containing a boronic acid.

According to another aspect, a system for administering a compound to anindividual is described. The system comprises, at least a polymercontaining a polyol and polymer containing a boronic acid capable ofreciprocal binding through a reversible covalent linkage, to beassembled in a nanoparticle herein described attaching the compound tobe administered to the individual according to methods herein described.

According to another aspect, a method to prepare a nanoparticlecomprising a polymer containing a polyol and a polymer containing aboronic acid is described. The method comprises contacting the polymercontaining polyols with the polymer containing a boronic acid for a timeand under condition to allow coupling of the polymer containing polyolywith the polymer containing a boronic acid.

According to another aspect, several polymer containing a boronic acidare described which are illustrated in details in the following sectionsof the present disclosure.

According to another aspect, several polymers containing polyols aredescribed, which are illustrated in details in the following sections ofthe present disclosure.

Also described herein are nanoparticles having a polymer containing apolyol that are conjugated to polymers having a nitrophenylboronic acidgroup, which enhances the stability of the nanoparticle by reducing itspKa.

Another aspect of the present disclosure provides a description oftargeted nanoparticles that, in some embodiments, can have only onesingle targeting ligand, which is capable of promoting delivery of thenanoparticle to a particular target, such as a cell expressing a bindingpartner for the targeting ligand of the particle. Targeted nanoparticlesof this sort have advantages over nanoparticles with a plurality oftargeting ligands, such as having a smaller overall size, due to havingfewer surface ligands, and have fewer ligands to mediate nonspecificbinding through avidity-based interactions, rather than affinity-basedinteractions. Additionally, nanoparticles that contain or carry atherapeutic agent can be successfully targeted to location of interest(such as a cell or tissue) using only a single targeting ligand, therebydelivering the therapeutic agent to the target at a very high targetingligand-to-therapeutic ratio. This aspect of the described nanoparticlescan significantly increase the efficiency of making such therapeuticswhile also reducing the need to employ a high number of costlyantibodies to mediate targeting.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments as a flexible molecularstructure suitable for carrying compounds of various sizes, dimensionsand chemical nature.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments as delivery systems which canprovide protection of the carried compound from degradation, recognitionby immune system and loss due to combination with serum proteins orblood cells.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments as delivery systemscharacterized by steric stabilization and/or ability to deliver thecompound to specific targets such as tissues, specific cell types withina tissue and even specific intracellular locations within certain celltypes.

Nanoparticles herein described and related compositions, methods, andsystems can be designed in several embodiments, to release a carriedcompound in a controllable way, including controlled release of multiplecompounds within a same nanoparticle at different rates and/or times.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments, to deliver compounds withenhanced specificity and/or selectivity during targeting and/or enhancedrecognition of the compound by the target compared to certain systems ofthe art.

Nanoparticles herein described and related compositions, methods, andsystems can be used in several embodiments in connection withapplications wherein controlled delivery of a compound of interest isdesirable, including but not limited to medical applications, such astherapeutics, diagnostics and clinical applications. Additionalapplications comprise biological analysis, veterinary applications, anddelivery of compounds of interest in organisms other than animals, andin particular in plants.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the detailed description and examplesbelow. Other features, objects, and advantages will be apparent from thedetailed description, examples and drawings, and from the appendedclaims

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description and theexamples, serve to explain the principles and implementations of thedisclosure.

FIG. 1 shows a schematic representation of a nanoparticle and a relatedmethod for the relevant formation in absence of a boronic acidcontaining compound. Panel A shows a schematic representation of apolymer containing a polyol (MAP, 4) and a compound of interest (nucleicacid) according to an embodiment herein described. Panel B shows ananoparticle formed upon assembly of the polymer containing a polyol andcompound shown in panel A.

FIG. 2 shows a schematic representation of a nanoparticle and a relatedmethod of manufacturing according to an embodiment of the presentdisclosure. Panel A shows a polymer containing a polyol (MAP, 4) and apolymer containing a boronic acid (BA-PEG, 6) together with a moleculeof interest (nucleic acid) according to an embodiment of the presentdisclosure. Panel B shows a BA-pegylated stabilized nanoparticle formedupon assembly of the polymers and compound shown in panel A.

FIG. 3 shows formation of a complex comprising polymers containingpolyols and a compound of interest according to an embodiment hereindescribed. In particular, FIG. 3, shows results of a MAP gel retardationassay with plasmid DNA according to an embodiment of the presentdisclosure. A DNA ladder is loaded in Lane 1. Lanes 2-8 show plasmid DNAcombined with MAP of incrementally increased charge ratio. Charge ratiois defined as the amount of positive charges on the MAP divided by theamount of negative charges on the nucleic acid.

FIG. 4 shows formation of a complex comprising polymers containingpolyols and a compound of interest according to an embodiment hereindescribed. In particular, FIG. 4 shows results of a MAP gel retardationassay with siRNA according to an embodiment of the present disclosure. ADNA ladder is loaded in Lane 1. Lanes 2-8 show siRNA combined with MAPof incrementally increased charge ratio.

FIG. 5 shows properties of nanoparticles according to some embodimentsherein described. In particular, FIG. 5 shows a diagram illustrating aplot of particle size (determined from dynamic light scattering (DLS)measurements) versus charge ratio and zeta potential (a property thatrelates to the surface charge of the nanoparticle) versus charge ratiofor MAP-plasmid nanoparticles according to an embodiment of the presentdisclosure.

FIG. 6 properties of nanoparticles according to some embodiments hereindescribed. In particular, FIG. 6 shows a diagram illustrating a plot ofparticle size (DLS) versus charge ratio and zeta potential versus chargeratio for BA-PEGylated MAP-plasmid nanoparticles according to anembodiment of the present disclosure.

FIG. 7 shows the salt stability of BA-PEGylated MAP-PlasmidNanoparticles according to an embodiment herein disclosed. Plot A: 5:1BA-PEG+np+1×PBS after 5 mins; Plot B: 5:1 BA-PEG+np, dialyzed 3× w/100kDa+1×PBS after 5 mins; Plot C: 5:1 prePEGylated w/BA-PEG+1×PBS after 5mins; Plot D: 5:1 prePEGylated w/BA-PEG, dialyze 3× w/100 kDa+PBS after5 mins.

FIG. 8 shows delivery of an agent to human cells in vitro withnanoparticles according to an embodiment herein described. Inparticular, FIG. 8 shows a diagram illustrating a plot of relative lightunits (RLU) that are a measure of the amount of luciferase proteinexpressed from the pGL3 plasmid that has been delivered to the cellsversus charge ratio for a MAP/pGL3 transfection into HeLa Cellsaccording to an embodiment of the present disclosure.

FIG. 9 shows delivery of an agent to a target with nanoparticlesaccording to an embodiment herein described. In particular, FIG. 9,shows a diagram illustrating a plot of cell survival versus charge ratioafter a MAP/pGL3 transfection according to an embodiment of the presentdisclosure. The survival data are for the experiments shown in FIG. 8.

FIG. 10 shows delivery of multiple compounds to a target withnanoparticles according to an embodiment herein described. Inparticular, FIG. 10 shows a diagram illustrating a plot of relativelight units (RLU) versus particle type for a co-transfection of MAPParticles containing pGL3 and siGL3 at a charge ratio of 5+/− into HeLaCells according to an embodiment herein described. The wording siCONindicates an siRNA with a control sequence.

FIG. 11 shows delivery of a compound to a target with nanoparticlesaccording to an embodiment herein described. In particular, FIG. 11shows a plot of relative light units (RLU) versus siGL3 concentrationfor a delivery of MAP/siGL3 at a charge ratio of 5+/− into HeLa-Luccells according to an embodiment of the present disclosure.

FIG. 12 shows a schematic representation of a synthesis of a polymercontaining a boronic acid presenting a targeting ligand according tosome embodiments herein described. In particular FIG. 12, show aschematic for a synthesis of boronic acid-PEG disulfide-Transferrinaccording to an embodiment of the present disclosure.

FIG. 13 shows a schematic representation of a synthesis of ananoparticle according to some embodiments herein described. Inparticular, FIG. 13 shows a schematic for a formulation of ananoparticle with Campothecin Mucic acid polymer (CPT-mucic acidpolymer) in water according to an embodiment of the present disclosure.

FIG. 14 shows a table summarizing particle sizes and zeta potentials ofnanoparticles formed from the CPT-mucic acid polymer conjugated inwater, prepared according to an embodiment of the present disclosure.

FIG. 15 shows a schematic representation of a synthesis of ananoparticle according to some embodiments herein described. Inparticular, FIG. 15 shows a formulation of a boronic acid-PEGylatednanoparticle with CPT-Mucic Acid Polymer and boronicacid-disulfide-PEG₅₀₀₀ in water according to an embodiment of thepresent disclosure.

FIG. 16 shows salt stability of MAP-4/siRNA stabilized with no PEG,nitroPEG-PEG and with 0.25 mol % Herceptin-PEG-nitroPBA. Formulated atcharge ratio 3 (+/−), 10×PBS was added at time 5 min such that theresulting solution was at 1×PBS.

FIGS. 17A and 17B show cellular uptake of (A) targeted MAP-CPTnanoparticles at increasing ratio of Herceptin®-PEG-nitroPBA tonanoparticle in BT-474, (B) medium MAP-CPT nanoparticles or targetedMAP-CPT nanoparticles with or without free Herceptin® (10 mg/ml) inBT-474 (shaded bars) and MCF-7 (open bars) cell lines.

FIGS. 18A and 18B show plasma pharmacokinetics of short, medium and longMAP-CPT nanoparticles and targeted MAP-CPT nanoparticles in BALB/c miceat 10 mg CPT/kg injections. Free CPT injected at 10 mg/kg into CD2F1mice is shown as comparison. (A) Plasma concentration of polymer boundCPT as a function of time. (B) Plasma concentration of unconjugated CPTas a function of time.

FIGS. 19A-D show the biodistribution of MAP-CPT (5 mg CPT/kg) andtargeted MAP-CPT (5 mg CPT/kg, 29 mg Herceptin®/kg) nanoparticles after4 and 24 h of treatment. (A) Percent injected dose (ID) of total CPT pergram of tumor, heart, liver, spleen, kidney or lung. (B) Percentage oftotal CPT in each organ that is unconjugated in tumor, heart, liver,spleen, kidney and lung. (C) Total concentration of CPT in plasma. (D)Percentage of total CPT that is unconjugated in plasma.

FIG. 20 depicts confocal immunofluorescence microscopy of BT-474 tumorsections taken from NCr nude mice treated with (A) MAP-CPT (5 mg CPT/kg)at 4 h (B) MAP-CPT (5 mg CPT/kg) at 24 h. (C) Targeted MAP-CPT (5 mgCPT/kg, 29 mg Herceptin®/kg) at 4 h. (D) Targeted MAP-CPT (5 mg CPT/kg,29 mg Herceptin®/kg) at 24 h. Left panel emission 440 nm (CPT, pink),center left panel emission 519 nm (MAP, green), center right panelemission 647 nm (Herceptin®, blue), right panel overlay of images.

FIG. 21 shows the antitumor efficacy study in NCr nude mice bearingBT-474 xenograft tumors. Mean tumor volume as a function time, groupscontaining Herceptin received 2 weekly doses, all other groups received3 weekly doses.

FIG. 22 shows the antitumor efficacy results for NCr nude mice treatedwith (A) Herceptin at 5.9 mg/kg. (B) Targeted MAP-CPT nanoparticles (5.9mg Herceptin®/kg and 1 mg CPT/kg).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Provided herein are nanoparticles and related compositions, methods, andsystems that can be used in connection for delivering a compound ofinterest (herein also cargo) comprised in the nanoparticles, methods forproducing the described nanoparticles, methods of treatment using thedescribed nanoparticles, and kits for assembling the describednanoparticles.

The term “nanoparticle” as used herein indicates a composite structureof nanoscale dimensions. In particular, nanoparticles are typicallyparticles of a size in the range of from about 1 to about 1000 nm, andare usually spherical although different morphologies are possibledepending on the nanoparticle composition. The portion of thenanoparticle contacting an environment external to the nanoparticle isgenerally identified as the surface of the nanoparticle. Innanoparticles herein described, the size limitation can be restricted totwo dimensions and so that nanoparticles herein described includecomposite structure having a diameter from about 1 to about 1000 nm,where the specific diameter depends on the nanoparticle composition andon the intended use of the nanoparticle according to the experimentaldesign. For example, nanoparticles to be used in several therapeuticapplications typically have a size of about 200 nm or below, and theones used, in particular, for delivery associated to cancer treatmenttypically have a diameter from about 1 to about 100 nm. The term“targeted nanoparticle” denotes a nanoparticle that is conjugated to atargeting agent or ligand.

Additional desirable properties of the nanoparticle, such as surfacecharges and steric stabilization, can also vary in view of the specificapplication of interest. Some of the exemplary properties that can bedesirable in clinical applications such as cancer treatment have beendescribed in the scientific literature. Additional properties areidentifiable by a skilled person upon reading of the present disclosure.Nanoparticle dimensions and properties can be detected by techniquesknown in the art. Exemplary techniques to detect particles dimensionsinclude but are not limited to dynamic light scattering (DLS) and avariety of microscopies such at transmission electron microscopy (TEM)and atomic force microscopy (AFM). Exemplary techniques to detectparticle morphology include but are not limited to TEM and AFM.Exemplary techniques to detect surface charges of the nanoparticleinclude but are not limited to zeta potential method. Additionaltechniques suitable to detect other chemical properties comprise by ¹H,¹¹B, and ¹³C and ¹⁹F NMR, UV/Vis and infrared/Raman spectroscopies andfluorescence spectroscopy (when nanoparticle is used in combination withfluorescent labels) and additional techniques identifiable by a skilledperson.

Nanoparticles and related compositions, methods, and systems hereindescribed can be used to deliver a compound of interest and inparticular an agent to a predetermined target.

The term “deliver” and “delivery” as used herein indicates the activityof affecting the spatial location of a compound, and in particularcontrolling said location. Accordingly, delivering a compound in thesense of the present disclosure indicates the ability to affectpositioning and movement of the compound at a certain time under acertain set of conditions, so that the compound's positioning andmovement under those conditions are altered with respect to thepositioning and movement that the compound would otherwise have.

In particular, delivery of a compound with respect to a referenceendpoint indicates the ability to control positioning and movement ofthe compound so that the compound is eventually positioned on theselected reference endpoint. In an in vitro system, delivery of acompound is usually associated to a corresponding modification of thechemical and/or biological detectable properties and activities of thecompound. In an in vivo system, delivery of a compound is also typicallyassociated with modification of the pharmacokinetics and possiblypharmacodynamics of the compound.

Pharmacokinetic of a compound indicates absorption, distribution,metabolism and excretion of the compound from the system, typicallyprovided by the body of an individual. In particular the term“absorption” indicates the process of a substance entering the body, theterm “distribution” indicates the dispersion or dissemination ofsubstances throughout the fluids and tissues of the body, the term“metabolism” indicates the irreversible transformation of parentcompounds into daughter metabolites and the term “excretion” indicatesthe elimination of the substances from the body. If the compound is in aformulation, pharmacokinetics also comprises liberation of the compoundfrom the formulation which indicates process of release of the compound,typically a drug, from the formulation. The term “pharmacodynamic”indicates physiological effects of a compound on the body or onmicroorganisms or parasites within or on the body and the mechanisms ofdrug action and the relationship between drug concentration and effect.A skilled person will be able to identify the techniques and proceduressuitable to detect pharmacokinetics and pharmacodynamic features andproperties of a compound of interest and in particular of an agent ofinterest such as a drug.

The term “agent” as used herein indicates a compound capable ofexhibiting a chemical or biological activity associated to the target.The term “chemical activity” as used herein indicates the ability of themolecule to perform a chemical reaction. The term biological activity asused herein indicates the ability of the molecule to affect a livingmatter. Exemplary chemical activities of agents comprise formation of acovalent or electrostatic interaction. Exemplary biological activitiesof agents comprise production and secretion of endogenous molecules,absorption and metabolization of endogenous or exogenous molecules andactivation or deactivation of genetic expression including transcriptionand translation of a gene of interest.

The term “target” as used herein indicates a biological system ofinterest including unicellular or pluricellular living organisms or anyportion thereof and include in vitro or in vivo biological systems orany portion thereof.

The nanoparticles herein described a polymer containing a boronic acidsis coupled to a polymer containing a polyol is arranged in thenanoparticle to be presented to an environment external to thenanoparticle.

The term a “polymer” as used herein indicates a large molecule composedof repeating structural units typically connected by covalent chemicalbonds. A suitable polymer may be a linear and/or branched, and can takethe form of a homopolymer or a co-polymer. If a co-polymer is used, theco-polymer may be a random copolymer or a branched co-polymer. Exemplarypolymers comprise water-dispersible and in particular water solublepolymers. For example, suitable polymers include, but are not limited topolysaccharides, polyesters, polyamides, polyethers, polycarbonates,polyacrylates, etc. For therapeutic and/or pharmaceutical uses andapplications, the polymer should have a low toxicity profile and inparticular that are not toxic or cytotoxic. Suitable polymers includepolymers having a molecular weight of about 500,000 or below. Inparticular, suitable polymers can have a molecular weight of about100,000 and below.

The term “polymer containing a polyol” or “polyol(s) polymer” as usedherein indicates a polymer presenting multiple hydroxyl functionalgroups. In particular, the polymer containing a polyol suitable to formthe nanoparticles here described comprise polymers presenting at least aportion of the hydroxyl functional groups for a coupling interactionwith at least one boronic acid of a polymer containing a boronic acid.

The term “present” as used herein with reference to a compound orfunctional group indicates attachment performed to maintain the chemicalreactivity of the compound or functional group as attached. Accordingly,a functional group presented on a surface, is able to perform under theappropriate conditions the one or more chemical reactions thatchemically characterize the functional group.

Structural units forming polymers containing polyols comprise monomericpolyols such as pentaerythritol, ethylene glycol and glycerin. Exemplarypolymers containing polyols comprise polyesters, polyethers andpolysaccharides. Exemplary suitable polyethers include but are notlimited to diols and in particular diols with the general formulaHO—(CH₂CH₂O)_(p)—H with p≧1, such as polyethylene glycol, polypropyleneglycol, and poly(tetramethylene ether) glycol. Exemplary, suitablepolysaccharides include but are not limited to cyclodextrins, starch,glycogen, cellulose, chitin and β-Glucans. Exemplary, suitablepolyesters include but are not limited to polycarbonate, polybutyrateand polyethylene terephthalate, all terminated with hydroxyl end groups.Exemplary polymers containing polyols comprise polymers of about 500,000or less molecular weight and in particular from about 300 to about100,000.

Several polymers containing polyols are commercially available and/orcan be produced using techniques and procedures identifiable by askilled person. Exemplary procedures for the synthesis of an exemplarypolyol polymer have been described previously in the scientificliterature, and others are illustrated in Examples 1-4. Additionalprocedures for making polymer containing polyols will be identifiable bya skilled person in view of the present disclosure.

The term “polymer containing a boronic acid” or “BA polymer” as usedherein indicates polymer containing at least one boronic acid grouppresented for binding to a hydroxyl group of a polymer containingpolyols. In particular, polymers containing boronic acids of thenanoparticles herein described include a polymer comprising in at leastone structural unit an alkyl or aryl substituted boronic acid containinga carbon to boron chemical bond. Suitable BA polymers comprise polymerswherein boronic acid is in a terminal structural unit or in any othersuitable position to provide the resulting polymer with hydrophilicproperties. Exemplary polymers containing polyols comprise polymers ofabout 40,000 or less molecular weight and in particular of about 20,000or less, or about 10,000 or less.

Several polymer containing a boronic acids are commercially availableand/or can be produced using techniques and procedures identifiable by askilled person. Exemplary procedures for the synthesis of an exemplarypolyol polymer have been described previously in the scientificliterature and other new ones are illustrated in Examples 5-8.Additional procedures for making BA polymers will be identifiable by askilled person in view of the present disclosure.

In the nanoparticles herein described polyols polymers are coupled tothe BA polymers. The term “coupled” or “coupling” as used herein withreference to attachment between two molecules indicates an interactionforming a reversible covalent linkage. In particular, in presence of asuitable medium, a boronic acid presented on the BA polymer interactwith hydroxyl groups of the polyols via a rapid and reversible pair-wisecovalent interaction to form boronic esters in a suitable medium.Suitable medium include water and several aqueous solutions andadditional organic media identifiable by a skilled person. Inparticular, when contacted in an aqueous medium BA polymers and polyolspolymers react, producing water as a side product. The boronic acidpolyol interaction is generally more favorable in aqueous solutions butis also known to proceed in organic media. In addition, cyclic estersformed with 1,2 and 1,3 diols are generally more stable than theiracyclic ester counterparts.

Accordingly, in a nanoparticle herein described, at least one boronicacid of the polymer containing a boronic acid is bound to hydroxylgroups of the polymer containing a polyol with a reversible covalentlinkage. Formation of a boronic ester between BA polymers and polyolspolymers can be detected by methods and techniques identifiable by askilled person such as boron-11 nuclear magnetic resonance (¹¹B NMR),potentiometric titration, UV/Vis and fluorescent detection techniqueswhereby the technique of choice is dependent on the specific chemicalnature and properties of the boronic acid and polyol composing thenanoparticle.

A nanoparticle resulting from coupling interactions of a BA polymerherein described with a polyol polymer herein described presents the BApolymer on the surface of the particle. In several embodiments thenanoparticles can have a diameter from about 1 to about 1000 nm and aspherical morphology although the dimensions and morphology of theparticle are largely determined by the specific BA polymer and polyolpolymers used to form the nanoparticles and by the compounds that arecarried on the nanoparticles according to the present disclosure.

In several embodiments the compound of interest carried by thenanoparticle forms part of the BA polymer and/or the polyol polymers.Examples of such embodiments are provided by nanoparticles wherein oneor more atoms of a polymer is replaced by a specific isotope e.g., ¹⁹Fand ¹⁰B, and are therefore suitable as agent for imaging the targetand/or providing radiation treatment to the target.

In several embodiments, the compound of interest carried thenanoparticle is attached to a polymer, typically a polyol polymer,through covalent or non-covalent linkage. Examples of such embodimentsare provided by nanoparticles wherein one or more moieties in at leastone of the polyol polymer and BA polymer attaches one or more compoundsof interest.

The term “attach”, “attached” or “attachment” as used herein, refers toconnecting or uniting by a bond, link, force or tie in order to keep twoor more components together, which encompasses either direct or indirectattachment such that for example where a first compound is directlybound to a second compound, and the embodiments wherein one or moreintermediate compounds, and in particular molecules, are disposedbetween the first compound and the second compound.

In particular, in some embodiments a compound can be attached to thepolyol polymer or BA polymer through covalent linkage of the compound tosuitable moieties of the polymer. Exemplary covalent linkages areillustrated in Example 19 where, attachment of the drug Camptothecin toMucic Acid polymer is performed through biodegradable ester bondlinkage, and in Example 9, wherein attachment of transferrin toBA-PEG₅₀₀₀ is performed through pegylation of the transferrin.

In some embodiments, the polymer can be designed or modified to enablethe attachment of a specific compound of interest, for example by addingone or more functional groups able to specifically bind a correspondingfunctional group on the compound of interest. For example, in severalembodiments it is possible to PEGylate the nanoparticle with a BA-PEG-X,where X can be a Maleimide or an iodoacetyl group or any leaving groupthat will react specifically with a thiol or non-specifically with anamine. The compound to be attached can then react to the maleimide oriodoacetyl groups after modification to express a thiol functionalgroup. The compound to be attached can also be modified with aldehydesor ketone groups and these can react via a condensation reaction withthe diols on the polyols to give acetals or ketals.

In some embodiments, a compound of interest can be attached to thepolyol polymer or BA polymer through non covalent bonds such as ionicbonds and intermolecular interactions, between a compound to be attachedand a suitable moiety of the polymer. Exemplary non covalent linkagesare illustrated in Example 10.

A compound of interest can be attached to the nanoparticle before, uponor after formation of the nanoparticle, for example via modification ofa polymer and/or of any attached compound in the particulate composite.Exemplary procedures to perform attachment of a compound on thenanoparticle are illustrated in the Examples section. Additionalprocedures to attach a compound to a BA polymer polyol polymer or othercomponents of the nanoparticle herein described (e.g. a previouslyintroduced compound of interest) can be identified by a skilled personupon reading of the present disclosure.

In some embodiments, at least one compound of interest attached to a BApolymer presented on the nanoparticle herein described is an agent thatcan be used as a targeting ligand. In particular, in severalembodiments, the nanoparticle attaches on the BA polymer one or moreagents to be used as a targeting ligand, and on the polyol polymerand/or the BA polymer, one or more agents to be delivered to a target ofchoice.

The term “targeting ligand” or “targeting agent” as used in the presentdisclosure indicates any molecule that can be presented on the surfaceof a nanoparticle for the purpose of engaging a specific target, and inparticular specific cellular recognition, for example by enabling cellreceptor attachment of the nanoparticle. Examples of suitable ligandsinclude, but are not limited to, vitamins (e.g. folic acid), proteins(e.g. transferrin, and monoclonal antibodies), monosaccharides (e.g.galactose), peptides, and polysaccharides. In particular targetingligands can be antibodies against certain surface cell receptors such asanti-VEGF, small molecules such as folic acid and other proteins such asholo-transferrin.

The choice of ligand, as one of ordinary skill appreciates, may varydepending upon the type of delivery desired. As another example, theligand may be membrane permeabilizing or membrane permeable agent suchas the TAT protein from HIV-1. The TAT protein is a viraltranscriptional activation that is actively imported into the cellnucleus. Torchilin, V. P. et al, PNAS. 98, 8786 8791, (2001). Suitabletargeting ligands attached to a BA polymer typically comprise a flexiblespacer such as a poly(ethylene oxide) with a boronic acid attached toits distal end (see Example 9).

In several embodiments, at least one of the compounds comprised orattached to the polyol polymer and/or BA polymer (including a targetingligand) can be an agent and in particular a drug, to be delivered to atarget, for example an individual, to which the chemical or biologicalactivity, e.g. the therapeutic activity, is to be exerted.

Selection of a polyol polymer and a BA polymer suitable to form ananoparticle herein described can be performed in view of the compoundand the target of interest. In particular, selection of a suitablepolymer containing a polyol and a suitable BA polymer to form ananoparticle herein described can be performed by providing candidatepolyol polymers and BA polymer, and selecting the polyol polymer and theBA polymer able to form a coupling interaction in the sense of thedisclosure, wherein the selected BA polymer and polyol polymer have achemical composition such that in view of the compound of interest andtargeting ligand to comprised or attached to the polyol polymers and/orthe BA polymers, the polyol polymers is less hydrophilic than the BApolymer. Detection of the BA polymer on the surface of the nanoparticleand related presentation on the environment external to the nanoparticlecan be performed by detection of the zeta potential which candemonstrate modification of the surface of the nanoparticle asillustrated in Example 12. (see in particular FIG. 6) Additionalprocedures to detect the surface charge of the particles and thestability of the particles in salt solutions, include detection ofchanges of the particle size such as the ones exemplified in Example 12(see in particular FIG. 7) and additional procedures identifiable by askilled person

In several embodiments, polymers containing polyols comprise one or moreof at least one of the following structural units

wherein

A is an organic moiety of formula

in which

-   -   R₁ and R₂ are independently selected from any carbon based or        organic group with a molecular weight of about 10 kDa or less;    -   X is independently selected from an aliphatic group, containing        one or more of —H, —F, —C, —N or —O; and    -   Y is independently selected from —OH or an organic moiety        bearing a hydroxyl (—OH) group including but not limited to        —CH₂OH, —CH₂CH₂OH, —CF₂OH, —CF₂CF₂OH, and C(R₁G₁)(RG₂)(R₁G₃)OH,        with R₁G₁, R₁G₂ and R₁G₃ are independently organic based        functionalities,        and

B is an organic moiety linking one of R₁ and R₂ of a first A moiety withone of the R₁ and R₂ of a second A moiety.

The term “moiety” as used herein indicates a group of atoms thatconstitute a portion of a larger molecule or molecular species. Inparticular, a moiety refers to a constituent of a repeated polymerstructural unit. Exemplary moieties include acid or base species,sugars, carbohydrates, alkyl groups, aryl groups and any other molecularconstituent useful in forming a polymer structural unit.

The term “organic moiety” as used herein indicates a moiety whichcontains a carbon atom. In particular, organic groups include naturaland synthetic compounds, and compounds including heteroatoms. Exemplarynatural organic moieties include but are not limited to most sugars,some alkaloids and terpenoids, carbohydrates, lipids and fatty acids,nucleic acids, proteins, peptides and amino acids, vitamins and fats andoils. Synthetic organic groups refer to compounds that are prepared byreaction with other compounds.

In several embodiments, one or more compounds of interest can beattached to (A), to (B) or to (A) and (B).

In several embodiments, R₁ and R₂ independently have the formula:

wherein

d is from 0 to 100

e is from 0 to 100

f is from 0 to 100,

Z is a covalent bond that links one organic moiety to another and inparticular to another moiety A or a moiety B as herein defined, and

Z₁ is independently selected from —NH₂, —OH, —SH, and —COOH

In several embodiments, Z can independently be selected from —NH—,—C(═O)NH—, —NH—C(═O), —SS—, —C(═O)O—, —NH(═NH₂ ⁺)— or —O—C(═O)—

In several embodiments where the structural unit A of a polymercontaining a polyol has formula (IV), X can be C_(v)H_(2v+1), wherev=0-5 and Y can be —OH

In some embodiments, R1 and/or R2 have formula (V) where Z is —NH(═NH₂⁺)— and/or Z₁ is NH₂.

In several embodiments, in polymers containing a polyol of the particleherein described (A) can be independently selected from the formulas

wherein

the spacer is independently selected from any organic moiety, and inparticular can include alkyl, phenyl or alkoxy groups optionallycontaining a heteroatom, such as sulfur, nitrogen, oxygen or fluorine;

the amino acid is selected from any organic group bearing a free amineand a free carboxylic acid group;

n is from 1 to 20; and

Z₁ is independently selected from —NH₂, —OH, —SH, and —COOH.

In several embodiments, Z1 is NH2, and/or the sugar can be anymonosaccharide such as glucose, fructose, mannitol, sucrose, galactose,sorbitol, xylose or galactose.

In several embodiments, in polymers containing a polyol of the particleherein described one ore more structural units (A) can independentlyhave the formula

In several embodiments, (B) can be formed by any straight, branched,symmetric or asymmetric compound linking the two (A) moieties throughfunctional groups.

In several embodiments, (B) can be formed by a compound where at leasttwo cross-linkable groups linking the two (A) moieties.

In some embodiments, (B) contains a neutral, cationic or anionic organicgroup whose nature and composition is dependent on the chemical natureof the compound to be covalently or non-covalently tethered

Exemplary cationic moieties of (B) for use with anionic cargo include,but are not limited to, organic groups bearing amidines groups,quartenary ammoniums, primary amine group, secondary amine group,tertiary amine groups (protonated below their pKa's), and immidazoliums

Exemplary anionic moieties contained in (B) for use with cationic cargoinclude, but are not limited to, organic groups bearing sulfonates offormula, nitrates of formula, carboxylates of formula, and phosphonates

In particular one or more cationic or anionic moieties (B) for use withanionic cargo and cationic cargos respectively can independently have ageneral formula of:

wherein R₅ is an electrophilic group that can be covalently linked to Awhen A contains nucleophilic groups. Examples of R₅ in this case includebut are not limited to —CO(═O)H, —C(═O)Cl, —C(═O)NHS, —C(═NH₂ ⁺)OMe,—S(═O)OCl—, —CH₂Br, alkyl and aromatic esters, terminal alkynes,tosylate, and mesylate amongst several others. In the case where moietyA contains electrophilic end groups, R₅ will bear nucleophilic groupssuch as —NH₂ (primary amines), —OH, —SH, N₃ and secondary amines.

In particular, when moiety (B) is a cationic moiety (B) for use withanionic cargo the “organic group” is an organic moiety that can have abackbone with a general formula consisting of C_(m)H_(2m) with m≧1 andother heteroatoms and must contain at least one of the followingfunctional groups including amidines of formula (XIII), quartenaryammoniums of formula (XIV), primary amine group of formula (XV),secondary amine group of formula (XVI), tertiary amine groups of formula(XVII) (protonated below their pKa's), and immidazoliums of formula(XVIII)

In embodiments, when moiety (B) is an anionic moiety (B) for use withcationic cargo, the “organic group” may have a backbone with a generalformula consisting of C_(m)H_(2m) with m≧1 and other heteroatoms andmust contain at least one of the following functional groups includingsulfonates of formula (XIX), nitrates of formula (XX), carboxylates offormula (XXI), and phosphonates of formula (XXII)

In embodiments wherein (B) is comprised by carboxylates (XXI), acompound containing primary amine or hydroxyl groups can also beattached via the formation of a peptide or an ester bond.

In embodiments wherein (B) is comprised of primary amine group offormula (XV), and/or secondary amine group of formula (XVI), a compoundcontaining carboxylic acid groups can also be attached via the formationof a peptide bond.

In several embodiments moiety (B) can independently be selected from

in which

-   -   q is from 1 to 20; and in particular can be 5    -   p is from 20 to 200; and    -   L is a leaving group.

The term “leaving group” as used herein indicates a molecular fragmentthat departs with a pair of electrons in heterolytic bond cleavage. Inparticular, a leaving group can be anions or neutral molecules, and theability of a leaving group to depart is correlated with the pK_(a) ofthe conjugate acid, with lower pK_(a) being associated with betterleaving group ability. Exemplary anionic leaving groups include halidessuch as Cl⁻, Br⁻, and I⁻, and sulfonate esters, such aspara-toluenesulfonate or “tosylate” (TsO⁻). Exemplary neutral moleculeleaving groups are water (H₂O), ammonia (NH₃), and alcohols (ROH).

In particular, in several embodiments, L can be a chloride (Cl), methoxy(OMe), t butoxy (OtBU) or N hydrosuccinimide (NHS).

In some embodiments the structural unit of formula (I) can have formula

In some embodiments the structural unit of formula (II) can have formula

In some embodiments the structural unit of formula (III) can haveformula

in which

n is from 1 to 20 and in particular from 1 to 4.

In some embodiments, the polymer containing polyol can have the formula

In some embodiments, the polymer containing a boronic acid contains atleast one terminal boronic acid group and has the following structure:

wherein

-   -   R₃ and R₄ can be independently selected from any hydrophilic        organic polymer, and in particular can independently be any        poly(ethylene oxides), and zwitterionic polymers.    -   X₁ can be an organic moiety containing one or more of —CH, —N,        or —B    -   Y₁ can be an alkyl group with a formula —C_(m)H_(2m)— with m≧1,        possibly containing olefins or alkynyl groups, or an aromatic        group such as a phenyl, biphenyl, napthyl or anthracenyl    -   r is from 1 to 1000,    -   a is from 0 to 3, and    -   b is from 0 to 3        and wherein functional group 1 and functional group 2 are the        same or different and are able to bind to a targeting ligand,        and in particular a protein, antibody or peptide, or is an end        group such as —OH, —OCH₃ or —(X₁)—(Y₁)—B(OH)₂—

In some embodiments, R₃ and R₄ are (CH₂CH₂O)_(t), where t is from 2 to2000 and in particular from 100 to 300

In some embodiments X₁ can be —NH—C(═O)—, —S—S—, —C(═O)—NH—, —O—C(═O)—or —C(═O)—O— and/or Y₁ can be a phenyl group.

In some embodiments r can be 1, a can be 0 and b can be 1.

In some embodiments, functional group 1 and functional group 2 are thesame or different and are independently selected from. —B(OH)₂, —OCH₃,—OH.

In particular, functional group 1 and/or 2 of formula (XXXI) can be afunctional group able to bind a cargo and in particular a targetingligand such as a protein, antibody or peptide, or can be an end groupsuch as —OH, —OCH₃ or —(X)—(Y)—B(OH)₂.

The term “functional group” as used herein indicates specific groups ofatoms within a molecular structure or portion thereof that areresponsible for the characteristic chemical reactions of that structureor portion thereof. Exemplary functional groups include hydrocarbons,groups containing halogen, groups containing oxygen, groups containingnitrogen and groups containing phosphorus and sulfur all identifiable bya skilled person. In particular, functional groups in the sense of thepresent disclosure include a carboxylic acid, amine, triarylphosphine,azide, acetylene, sulfonyl azide, thio acid and aldehyde. In particular,for example, a functional group able to bind a corresponding functionalgroup in a targeting ligand can be selected to comprise the followingbinding partners: carboxylic acid group and amine group, azide andacetylene groups, azide and triarylphosphine group, sulfonyl azide andthio acid, and aldehyde and primary amine. Additional functional groupscan be identified by a skilled person upon reading of the presentdisclosure. As used herein, the term “corresponding functional group”refers to a functional group that can react to another functional group.Thus, functional groups that can react with each other can be referredto as corresponding functional groups.

An end-group indicates a constitutional unit that is an extremity of amacromolecule or oligomer molecule. For example the end-group of a PETpolyester may be an alcohol group or a carboxylic acid group. End groupscan be used to determine molar mass. Exemplary end groups comprise —OH.—COOH, NH₂, and OCH₃,

In some embodiments, the polymer containing boronic acid can haveformula

wherein s is from 20 to 300.

Exemplary agents and targeting ligands that can be attached tonanoparticles of the present disclosure comprise organic or inorganicmolecules, including polynucleotides, nucleotides, aptamerspolypeptides, proteins, polysaccharides macromolecular complexesincluding but not limited to those comprising a mixture of protein andpolynucleotides, saccharides and/or polysaccharides, viruses, moleculeswith radioisotopes, antibodies or antibody fragments.

The term “polynucleotide” as used herein indicates an organic polymercomposed of two or more monomers including nucleotides, nucleosides oranalogs thereof. The term “nucleotide” refers to any of severalcompounds that consist of a ribose or deoxyribose sugar joined to apurine or pyrimidine base and to a phosphate group and that is the basicstructural unit of nucleic acids. The term “nucleoside” refers to acompound (such as guanosine or adenosine) that consists of a purine orpyrimidine base combined with deoxyribose or ribose and is foundespecially in nucleic acids. The term “nucleotide analog” or “nucleosideanalog” refers respectively to a nucleotide or nucleoside in which oneor more individual atoms have been replaced with a different atom or awith a different functional group. Accordingly, the term“polynucleotide” includes nucleic acids of any length, and in particularDNA, RNA, analogs and fragments thereof. A polynucleotide of three ormore nucleotides is also called “nucleotidic oligomer” or“oligonucleotide.”

The term “aptamers” as used here indicates oligonucleic acid or peptidemolecules that bind a specific target. In particular, nucleic acidaptamers can comprise, for example, nucleic acid species that have beenengineered through repeated rounds of in vitro selection orequivalently, SELEX (systematic evolution of ligands by exponentialenrichment) to bind to various molecular targets such as smallmolecules, proteins, nucleic acids, and even cells, tissues andorganisms. Aptamers are useful in biotechnological and therapeuticapplications as they offer molecular recognition properties that rivalthat of the antibodies. Peptide aptamers are peptides that are designedto specifically bind to and interfere with protein-protein interactionsinside cells. In particular, peptide aptamers can be derived, forexample, according to a selection strategy that is derived from theyeast two-hybrid (Y2H) system. In particular, according to thisstrategy, a variable peptide aptamer loop attached to a transcriptionfactor binding domain is screened against the target protein attached toa transcription factor activating domain. In vivo binding of the peptideaptamer to its target via this selection strategy is detected asexpression of a downstream yeast marker gene.

The term “polypeptide” as used herein indicates an organic linear,circular, or branched polymer composed of two or more amino acidmonomers and/or analogs thereof. The term “polypeptide” includes aminoacid polymers of any length including full length proteins and peptides,as well as analogs and fragments thereof. A polypeptide of three or moreamino acids is also called a protein oligomer, peptide or oligopeptide.In particular, the terms “peptide” and “oligopeptide” usually indicate apolypeptide with less than 50 amino acid monomers. As used herein theterm “amino acid”, “amino acidic monomer”, or “amino acid residue”refers to any of the twenty naturally occurring amino acids, non-naturalamino acids, and artificial amino acids and includes both D an L opticalisomers. In particular, non-natural amino acids include D-stereoisomersof naturally occurring amino acids (these including useful ligandbuilding blocks because they are not susceptible to enzymaticdegradation). The term “artificial amino acids” indicate molecules thatcan be readily coupled together using standard amino acid couplingchemistry, but with molecular structures that do not resemble thenaturally occurring amino acids. The term “amino acid analog” refers toan amino acid in which one or more individual atoms have been replaced,either with a different atom, isotope, or with a different functionalgroup but is otherwise identical to original amino acid from which theanalog is derived. All of these amino acids can be syntheticallyincorporated into a peptide or polypeptide using standard amino acidcoupling chemistries. The term “polypeptide” as used herein includespolymers comprising one or more monomer, or building blocks other thanan amino acid monomer. The terms monomer, subunit, or building blocksindicate chemical compounds that under appropriate conditions can becomechemically bonded to another monomer of the same or different chemicalnature to form a polymer. The term “polypeptide” is further intended tocomprise a polymer wherein one or more of the building blocks iscovalently bound to another by a chemical bond other than amide orpeptide bond.

The term “protein” as used herein indicates a polypeptide with aparticular secondary and tertiary structure that can participate in, butnot limited to, interactions with other biomolecules including otherproteins, DNA, RNA, lipids, metabolites, hormones, chemokines, and smallmolecules. Exemplary proteins herein described are antibodies.

The term “antibody” as used herein refers to a protein of the kind thatis produced by activated B cells after stimulation by an antigen and canbind specifically to the antigen promoting an immune response inbiological systems. Full antibodies typically consist of four subunitsincluding two heavy chains and two light chains. The term antibodyincludes natural and synthetic antibodies, including but not limited tomonoclonal antibodies, polyclonal antibodies or fragments thereof.Exemplary antibodies include IgA, IgD, IgG1, IgG2, IgG3, IgM and thelike. Exemplary fragments include Fab Fv, Fab′ F(ab′)2 and the like. Amonoclonal antibody is an antibody that specifically binds to and isthereby defined as complementary to a single particular spatial andpolar organization of another biomolecule which is termed an “epitope”.In some forms, monoclonal antibodies can also have the same structure. Apolyclonal antibody refers to a mixture of different monoclonalantibodies. In some forms, polyclonal antibodies can be a mixture ofmonoclonal antibodies where at least two of the monoclonal antibodiesbinding to a different antigenic epitope. The different antigenicepitopes can be on the same target, different targets, or a combination.Antibodies can be prepared by techniques that are well known in the art,such as immunization of a host and collection of sera (polyclonal) or bypreparing continuous hybridoma cell lines and collecting the secretedprotein (monoclonal).

In several embodiments, polyol polymers form a non-covalent complex orlinkage with one or more compounds of interest to be delivered accordingto the schematic illustration of FIGS. 1 and 2.

In several embodiments, a nanoparticle structure comprises an agent anda polymer containing a polyol, where the agent is linked to a polyolpolymer by a covalent bond. An example of a polyol polymer conjugated toan agent is detailed in Examples 16-21. In these embodiments, polyolpolymers conjugated to an agent (herein referred to as “polyolpolymer-agent conjugate”) form nanoparticles whose structure presentssites on their surface for interaction with BA molecules.

In several of those embodiments, the nanoparticle further comprises BApolymers configured to provide steric stabilization and/or targetingfunctionality to the nanoparticle. In particular, in those embodimentsthe addition of a BA polymer allows minimizing of self-aggregation andundesired interactions with other nanoparticles, thus providing enhancedsalt and serum stability. For example, steric stabilization is hereinprovided by the BA polymer having PEG as illustrated by the exemplarynanoparticle described in Example 12.

In such embodiments, the structure of this nanoparticle affords severaladvantages over agents delivery methods of the prior art, such as theability to provide controlled release of one or more agents. Thisfeature can be provided, for example, by the use of a biodegradableester linkage between the agent and the polyol polymer. A person skilledin the art will recognize other potential linkages suitable for thispurpose. In these embodiments, another advantage is the ability toprovide specific targeting of the agent through the BA polymer moiety.

In several embodiments, BA polymers may comprise a fluorinated boronicacid (Example 7) or a fluorinated cleavable boronic acid (Example 8)capable of being used as an imaging agent in MRI or other similartechniques. Such an imaging agent may be useful for tracking thepharmacokinetics or pharmacodynamics of an agent delivered by thenanoparticle.

In several embodiments, a nanoparticle structure comprises an agent anda polyol polymer, where the nanoparticle is a modified liposome. Inthese embodiments, the modified liposome comprises lipids conjugated topolyol polymers via a covalent linkage such that the surface of theliposome presents polyol polymers. In these embodiments, the modifiedliposomes form such that the agents to be delivered are contained withinthe liposome nanoparticle.

The term “liposome” as used herein indicates a vesicular structurecomprised of lipids. The lipids typically have a tail group comprising along hydrocarbon chain and a hydrophilic head group. The lipids arearranged to form a lipid bilayer with an inner aqueous environmentsuitable to contain an agent to be delivered. Such liposomes present anouter surface that may comprise suitable targeting ligands or moleculesfor specific recognition by cell surface receptors or other targets ofinterest.

The term “conjugated” as used herein indicates that one molecule hasformed a covalent bond with a second molecule and includes linkageswhere atoms covalently bond with alternating single and multiple (e.g.double) bonds (e.g., C═C—C═C—C) and influence each other to produceelectron delocalization.

In yet other embodiments of the present disclosure, a nanoparticlestructure comprises an agent and a polyol, where the nanoparticle is amodified micelle. In these embodiments, the modified micelle comprisespolyol polymers modified to contain a hydrophobic polymer block.

The term “hydrophobic polymer block” as used in the present disclosureindicates a segment of the polymer that on its own would be hydrophobic.

The term “micelle” as used herein refers to an aggregate of moleculesdispersed in a liquid. A typical micelle in aqueous solution forms anaggregate with the hydrophilic “head” regions in contact withsurrounding solvent, sequestering the hydrophobic single tail regions inthe micelle centre. In the present disclosure the head region may be,for example, a surface region of the polyol polymer while the tailregion may be, for example, the hydrophobic polymer block region of thepolyol polymer.

In these embodiments, polyol polymers with a hydrophobic polymer block,when mixed with an agent to be delivered, arrange to form a nanoparticlethat is a modified micelle with agents to be delivered contained withinthe nanoparticle. Such nanoparticle embodiments present polyol polymerson their surface that are suitable to interact with BA polymers that door do not have targeting functionality according to previousembodiments. In these embodiments, BA polymers capable of use for thispurpose include those with hydrophilic A and hydrophobic B in formula(I) or (II). This interaction provides the same or similar advantages asit does for other nanoparticle embodiments mentioned above.

In some embodiments, nanoparticles or related components can becomprised in a composition together with an acceptable vehicle. The term“vehicle” as used herein indicates any of various media acting usuallyas solvents, carriers, binders, excipients or diluents for ananoparticle comprised in the composition as an active ingredient.

In some embodiments, where the composition is to be administered to anindividual the composition can be a pharmaceutical composition and theacceptable vehicle can be a pharmaceutically acceptable vehicle.

In some embodiments, a nanoparticle can be included in pharmaceuticalcompositions together with an excipient or diluent. In particular, insome embodiments, pharmaceutical compositions are disclosed whichcontain nanoparticle, in combination with one or more compatible andpharmaceutically acceptable vehicle, and in particular withpharmaceutically acceptable diluents or excipients.

The term “excipient” as used herein indicates an inactive substance usedas a carrier for the active ingredients of a medication. Suitableexcipients for the pharmaceutical compositions herein disclosed includeany substance that enhances the ability of the body of an individual toabsorb the nanoparticle. Suitable excipients also include any substancethat can be used to bulk up formulations with nanoparticles to allow forconvenient and accurate dosage. In addition to their use in thesingle-dosage quantity, excipients can be used in the manufacturingprocess to aid in the handling of nanoparticles. Depending on the routeof administration, and form of medication, different excipients may beused. Exemplary excipients include but are not limited to antiadherents,binders, coatings disintegrants, fillers, flavors (such as sweeteners)and colors, glidants, lubricants, preservatives, sorbents.

The term “diluent” as used herein indicates a diluting agent which isissued to dilute or carry an active ingredient of a composition.Suitable diluents include any substance that can decrease the viscosityof a medicinal preparation.

In certain embodiments, compositions and, in particular, pharmaceuticalcompositions can be formulated for systemic administration, whichincludes parenteral administration and more particularly intravenous,intradermic, and intramuscular administration.

Exemplary compositions for parenteral administration include but are notlimited to sterile aqueous solutions, injectable solutions orsuspensions including nanoparticles. In some embodiments, a compositionfor parenteral administration can be prepared at the time of use bydissolving a powdered composition, previously prepared in a freeze-driedlyophilized form, in a biologically compatible aqueous liquid (distilledwater, physiological solution or other aqueous solution).

The term “lyophilization” (also known as freeze-drying orcryodesiccation) indicates a dehydration process typically used topreserve a perishable material or make the material more convenient fortransport. Freeze-drying works by freezing the material and thenreducing the surrounding pressure and adding enough heat to allow thefrozen water in the material to sublime directly from the solid phase togas.

In pharmaceutical applications freeze-drying is often used to increasethe shelf life of products, such as vaccines and other injectables. Byremoving the water from the material and sealing the material in a vial,the material can be easily stored, shipped, and later reconstituted toits original form for injection.

In several embodiments nanoparticles herein described are delivered to apredetermined target. In some embodiments, the target is an in vitrobiological system and the method comprises contacting target with thenanoparticle herein described.

In some embodiments, a method is provided for delivery of an agent to anindividual where the method comprises formulating a suitablenanoparticle according to various disclosed embodiments. Thenanoparticles may also be formulated into a pharmaceutically acceptablecomposition according to several disclosed embodiments. The methodfurther comprises delivering a nanoparticle to a subject. To deliver thenanoparticle to an individual, the nanoparticle or nanoparticleformulations may be given orally, parenterally, topically, or rectally.They are delivered in forms suitable for each administration route. Forexample, nanoparticle compositions can be administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories.

The term “individual” as used herein includes a single biologicalorganism including but not limited to plants or animals and inparticular higher animals and in particular vertebrates such as mammalsand in particular human beings.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal, injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a nanoparticle or composition thereofother than directly into the central nervous system, such that it entersthe individual's system and, thus, is subject to metabolism and otherlike processes, for example, subcutaneous administration.

Actual dosage levels of the active ingredient or agent in thepharmaceutical compositions herein described may be varied so as toobtain an amount of the active ingredient which is effective to achievethe desired therapeutic response for a particular individual,composition, and mode of administration, without being toxic to theindividual.

These therapeutic polymer conjugate may be administered to humans andother animals for therapy by any suitable route of administration,including orally, nasally, as by, for example, a spray, rectally,intravaginally, parenterally, intracisternally and topically, as bypowders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the therapeuticpolymer conjugate, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

In particular in some embodiments, the compound delivered is a drug fortreating or preventing a condition in the individual.

The term “drug” or “therapeutic agent” indicates an active agent thatcan be used in the treatment, prevention, or diagnosis of a condition inthe individual or used to otherwise enhance the individual's physical ormental well-being.

The term “condition” as used herein indicates a usually the physicalstatus of the body of an individual, as a whole or of one or more of itsparts, that does not conform to a physical status of the individual, asa whole or of one or more of its parts, that is associated with a stateof complete physical, mental and possibly social well-being. Conditionsherein described include but are not limited disorders and diseaseswherein the term “disorder” indicates a condition of the livingindividual that is associated to a functional abnormality of the body orof any of its parts, and the term “disease” indicates a condition of theliving individual that impairs normal functioning of the body or of anyof its parts and is typically manifested by distinguishing signs andsymptoms. Exemplary conditions include but are not limited to injuries,disabilities, disorders (including mental and physical disorders),syndromes, infections, deviant behaviors of the individual and atypicalvariations of structure and functions of the body of an individual orparts thereof.

The term “treatment” as used herein indicates any activity that is partof a medical care for or deals with a condition medically or surgically.

The term “prevention” as used herein indicates any activity, whichreduces the burden of mortality or morbidity from a condition in anindividual. This takes place at primary, secondary and tertiaryprevention levels, wherein: a) primary prevention avoids the developmentof a disease; b) secondary prevention activities are aimed at earlydisease treatment, thereby increasing opportunities for interventions toprevent progression of the disease and emergence of symptoms; and c)tertiary prevention reduces the negative impact of an alreadyestablished disease by restoring function and reducing disease-relatedcomplications.

Exemplary compounds that can be delivered by the nanoparticles hereindescribed and that are suitable as drugs comprise compounds able to emitelectromagnetic radiations (such as ¹⁰B isotopes) to be used inradiation treatments (such as boron neutron capture) Additionaltherapeutic agents comprise any lipophilic or hydrophilic, synthetic ornaturally occurring biologically active therapeutic agent includingthose known in the art. The Merck Index, An Encyclopedia of Chemicals,Drugs, and Biologicals, 13th Edition, 2001, Merck and Co., Inc.,Whitehouse Station, N.J. Examples of such therapeutic agents include,but are not limited to, small molecule pharmaceuticals, antibiotics,steroids, polynucleotides (e.g. genomic DNA, cDNA, mRNA, siRNA, shRNA,miRNA, antisense oligonucleotides, viruses, and chimericpolynucleotides), plasmids, peptides, peptide fragments, small molecules(e.g. doxorubicin), chelating agents (e.g. deferoxamine (DESFERAL),ethylenediaminetetraacetic acid (EDTA)), natural products (e.g. Taxol,Amphotericin), and other biologically active macromolecules such as, forexample, proteins and enzymes. See also U.S. Pat. No. 6,048,736 whichlists active agents (therapeutic agents) that can be used as therapeuticagent with nanoparticles herein described. Small molecule therapeuticagents may not only be the therapeutic agent within the compositeparticle but, in an additional embodiment, may be covalently bound to apolymer in the composite. In several embodiments, the covalent bond isreversible (e.g. through a prodrug form or biodegradable linkage such asa disulfide) and provides another way of delivering the therapeuticagent. In several embodiments therapeutic agent that can be deliveredwith the nanoparticles herein described include chemotherapeutics suchas epothilones, camptothecin-based drugs, taxol, or a nucleic acid suchas a plasmid, siRNA, shRNA, miRNA, antisense oligonucleotides aptamersor their combination, and additional drugs identifiable by a skilledperson upon reading of the present disclosure.

In some embodiments, the compound delivered is a compound suitable forimaging a cell or tissue of the individual. Exemplary compounds that canbe delivered by the nanoparticles herein described and that are suitablefor imaging comprise compounds that contain isotopes such as ¹⁹Fisotopes for MR imaging, ¹⁸F or ⁶⁴Cu for PET imaging etc.

In particular, the nanoparticles described herein can be configured tocontain ¹⁹F-containing BA polymers. For example, ¹⁹F atoms can beincorporated into a non-cleavable or cleavable BA polymer compound.Other locations for the ¹⁹F atoms are possible on the BA polymercomponent, the polyol polymer component, or on the agent to bedelivered. These and other variations will be apparent to one skilled inthe art.

In several embodiments, the nanoparticles herein described can be usedto deliver chemicals used in the agricultural industry. In anotherembodiment of the invention, the agent delivered by the nanoparticleherein described is a biologically active compound having microbiocidaland agricultural utility. These biologically active compounds includethose known in the art. For example, suitable agriculturallybiologically active compounds include, but are not limited to,fertilizers, fungicides, herbicides, insecticides, and mildewcides.Microbicides are also used in water-treatment to treat municipal watersupplies and industrial water systems such as cooling waters, whitewater systems in papermaking Aqueous systems susceptible tomicrobiological attack or degradation are also found in the leatherindustry, the textile industry, and the coating or paint industry.Examples of such microbicides and their uses are described, individuallyand in combinations, in U.S. Pat. Nos. 5,693,631, 6,034,081, and6,060,466, which are incorporated herein by reference. Compositionscontaining active agents such as those discussed above may be used inthe same manner as known for the active ingredient itself. Notably,because such uses are not pharmacological uses, the polymer of thecomposite does not necessarily have to meet the toxicity profilerequired in pharmaceutical uses.

In certain embodiments, nanoparticles comprising polyol polymers and BApolymers can also be comprised in a system suitable for delivering anyof the compounds herein indicated and in particular agents, using ananoparticle. In some embodiments of the system, nanoparticles areprovided with components suitable for preparing the nanoparticles foradministration to an individual.

The systems herein disclosed can be provided in the form of kits ofparts. For example the polyol polymers and/or BA polymers can beincluded as a molecule alone or in the presence of suitable excipients,vehicles or diluents.

In a kit of parts, polyol polymers, BA polymers, and/or agents to bedelivered are comprised in the kit independently possibly included in acomposition together with suitable vehicle carrier or auxiliary agents.For example, polyol polymers and/or BA polymers can be included in oneor more compositions alone and/or included in a suitable vector. Also,an agent to be delivered can be included in a composition together witha suitable vehicle carrier or auxiliary agent. Alternatively, the agentmay be supplied by the end user and may be absent from the kit of parts.Furthermore, the polyol polymers, BA polymers and/or agents can beincluded in various forms suitable for appropriate incorporation into ananoparticle.

Additional components can also be included and comprise microfluidicchip, reference standards, buffers, and additional componentsidentifiable by a skilled person upon reading of the present disclosure.

In the kit of parts herein disclosed, the components of the kit can beprovided, with suitable instructions and other necessary reagents, inorder to perform the methods here disclosed. In some embodiments, thekit can contain the compositions in separate containers. Instructions,for example written or audio instructions, on paper or electronicsupport such as tapes or CD-ROMs, for carrying out the assay, can alsobe included in the kit. The kit can also contain, depending on theparticular method used, other packaged reagents and materials (such aswash buffers and the like).

Further details concerning the identification of the suitable carrieragent or auxiliary agent of the compositions, and generallymanufacturing and packaging of the kit, can be identified by the personskilled in the art upon reading of the present disclosure.

In some embodiments, a nanoparticle may be prepared by preparing theindividual components of the nanoparticle followed by mixing thecomponents in various orders to arrive at a desired compositenanoparticle structure. Preparation and mixing of components is carriedout in suitable solutions known by those skilled in the art.

The term “mixing” as used herein indicates addition of one solutioncomprising a molecule of interest with another solution comprisinganother molecule of interest. For example, an aqueous solution of polyolpolymers may be mixed with an aqueous solution of BA polymers in thecontext of the present disclosure.

The term “solution” as used herein indicates any liquid phase samplecontaining molecules of interest. For example, an aqueous solution ofpolyol polymers may comprise polyol polymers diluted in water or anybuffered solution, in particular aqueous solutions.

In some embodiments, a nanoparticle can be prepared by mixing polyolpolymers with an agent to be delivered (FIGS. 1 and 2), forming a polyolpolymer-agent nanoparticle. In other embodiments, a nanoparticle may beprepared by further mixing BA polymers with the polyol polymer-agentnanoparticle. In other embodiments, a nanoparticle is prepared by mixingpolyol polymers with BA polymers, followed by mixing an agent to bedelivered. In yet other embodiments, a nanoparticle is prepared bysimultaneously mixing polyol polymers, BA polymers, and an agent to bedelivered.

In some embodiments, a nanoparticle is prepared by forming a polyolpolymer-agent conjugate according to various embodiments of the presentdisclosure, thus preparing a nanoparticle comprised of a polyolpolymer-agent conjugate. In other embodiments nanoparticles comprised ofa polyol polymer-agent conjugates may be prepared by dissolving thenanoparticles in a suitable aqueous solution. In yet furtherembodiments, nanoparticles comprised of a polyol polymer-agentconjugates may be prepared by mixing polyol polymer-agent conjugateswith BA polymers that do or do not provide targeting ligand.

In some embodiments, a nanoparticle can be prepared by mixing polyolpolymers with a hydrophobic polymer block with an agent to be delivered,thus preparing a modified micelle according to embodiments of thepresent disclosure. In other embodiments, a nanoparticle may be preparedby further mixing the modified micelle with BA polymers. In yet otherembodiments, a nanoparticle may be prepared by mixing polyol polymerswith BA polymers, followed by mixing an agent to be delivered, thuspreparing a nanoparticle that is a modified micelle.

In some embodiments of the present disclosure, a nanoparticle can beprepared by mixing lipids conjugated with polyol polymers with BApolymers and/or agents to be delivered, thus preparing a modifiedliposome. In various embodiments, a nanoparticle may be prepared bymixing lipids conjugated with polyol polymers with BA polymers, followedby mixing agents to be delivered. In other embodiments, a nanoparticlemay be prepared by mixing lipids conjugated with polyol polymers withagents to be delivered. In other embodiments, a nanoparticle may beprepared by mixing lipids conjugated with polyol polymers with agents tobe delivered, followed by mixing BA polymers, thus preparing ananoparticle that is a modified liposome.

The formation of nanoparticles according to several embodiments of thepresent disclosure can be analyzed with techniques and procedures knownby those with skill in the art. For example, in several embodiments, gelretardation assays are used to monitor and measure the incorporation ofa nucleic acid agent within a nanoparticle (Example 10). In severalembodiments, a suitable nanoparticle size and/or zeta potential can bechosen using known methods (Example 11).

Further details concerning the identification of the suitable carrieragent or auxiliary agent of the compositions, and generallymanufacturing and packaging of the kit, can be identified by the personskilled in the art upon reading of the present disclosure.

Nanoparticles Having a Polymer with Nitrophenylboronic Acid

Described herein are nanoparticles having a polymer containing a polyolthat is conjugated to a polymer containing a nitrophenylboronic acid.The polymer containing a polyol nanoparticle segment of the targetednanoparticles described can have one or more of any one of the followingstructural units:

where A is an organic moiety of formula

in which R₁ and R₂ are independently selected from any carbon-based ororganic group with a molecular weight of about 10 kDa or less; X isindependently selected from an aliphatic group containing one or more of—H, —F, —C, —N or —O; and Y is independently selected from —OH or anorganic moiety presenting an —OH, and B is an organic moiety linking oneof the R₁ and R₂ of a first moiety A with one of the R₁ and R₂ of asecond moiety A in the polymer. In some embodiments X can beC_(n)H_(2n+1), in which n is any single number from 0-5 and Y is —OH. Insome embodiments A can be any one of:

where the spacer is independently selected from any organic group; theamino acid is selected from any organic group bearing a free amine and afree carboxylic acid group; n is any single number from 1 to 20; and Z₁is independently selected from —NH₂, —OH, —SH, and —COOH; R₁ and R₂independently can have the formula:

wherein d is any single number from 0 to 100, e is any single numberfrom 0 to 100, f is any single number from 0 to 100, Z is a covalentbond linking one organic moiety to another, and Z₁ is independentlyselected from —NH₂, —OH, —SH, and —COOH; B can be any one of

in which q is any single number from 1-20; p is any single number from20-200; and L is a leaving group, where these B subunits are paired withany one of the A subunits described above. In more particularembodiments, the polymer containing a polyol nanoparticle segment of thetargeted nanoparticles shown in structural unit of formula (I) can be:

the polymer containing a polyol nanoparticle segment of the targetednanoparticles shown instructural unit of formula (II) can be:

andthe polymer containing a polyol nanoparticle segment of the targetednanoparticles shown in structural unit of formula (III) can be:

in which n is any single number from 1-20. In some embodiments of thedescribed targeted nanoparticle, the polymer containing a polyol is:

In summary, any one of the formulas for subpart A (formula VI, VII, orVIII) can be combined with any one of the formulas for subpart B(formula XXIII or XIV) to form the polymer containing a polyol of thedescribed nanoparticles. In certain aspects described herein thenanoparticles can have a polymer containing a polyol formed from thecombination of anyone of the formulas for subpart A (IX, X, or XI) withany one of the formulas for subpart B (formula XXIII or XIV).

In some embodiments the polymer containing a nitrophenylboronic acidcomprises a nitrophenylboronic acid group and has the general formula:

where R₃ and R₄ are independently an hydrophilic organic polymer, X₁ isan organic moiety containing one or more of —C, —N, or —B, Y₁ is analkyl group of formula —C_(m)H_(2m)—, in which m is ≧1 or an aromaticgroup, r is any single number from 1-1000, a is any single number from0-3, and b is any single number from 0-3 and functional group 1 andfunctional group 2 may be the same or different and may be independentlyselected from any one of —B(OH)₂, —OCH₃, or —OH. In some embodimentsthese variable subparts of the described polymer containing anitrophenylboronic acid can be selected from the following: R₃ and R₄may be (CH₂CH₂O)_(t), where t is any single number from 2 to 2000; X₁ isany one of —NH—C(═O)—, —S—S—, —C(═O)—NH—, —O—C(═O)— or —C(═O)—O— and Y₁is a nitrophenyl group. In some embodiments these variable subparts ofthe described polymer containing a nitrophenylboronic acid can beselected from the following: R₃ and R₄ may be (CH₂CH₂O)_(t), where t isany single number from 2 to 2000; X₁ is any one of —NH—C(═O)—, —S—S—,—C(═O)—NH—, —O—C(═O)— or —C(═O)—O— and Y₁ is a nitrophenyl group, and rcan have a value of 1, a can have a value of 0 and b can have a valueof 1. In each of the embodiments of the polymer containing anitrophenylboronic acid, the nitro group can be at either the ortho,meta, or para position, relative to the boronic acid group, of thephenyl ring. In still further embodiments, the polymer containing anitrophenylboronic acid can have additional groups present on the phenylring, such as a methyl group. In a particular embodiment the targetednanoparticle of described herein can include a polymer containing anitrophenylboronic acid having any one of the following formulas:

where s is any single number from 20-300. The polymers of formulasXXXIII, XXXIV, and XXXV can be further modified to change the positionof the PEG on the phenyl ring to be in the ortho, meta, or para positionrelative to the boronic acid group.

In summary, any one of the formulas for subpart A (formula VI, VII, orVIII) can be combined with any one of the formulas for subpart B(formula XXIII or XIV) to form the polymer containing a polyolnanoparticle segment of the described nanoparticles, the resultingpolymer containing a polyol can then be coupled to a polymer containinga nitrophenylboronic acid. In some embodiments the conjugation betweenthe described polymer containing a polyol and the described polymercontaining a nitrophenylboronic acid will be mediated by at least onehydroxyl group of the boronic acid group. In certain aspects describedherein the nanoparticles can have a polymer containing a polyol formedfrom the combination of anyone of the formulas for subpart A (IX, X, orXI) with any one of the formulas for subpart B (formula XXIII or XIV),which can then be coupled to a polymer containing a boronic acid havingformula XXX. In some embodiments described herein, the nanoparticles canhave a polymer containing a polyol formed from the combination of anyoneof the formulas for subpart A (IX, X, or XI) with any one of theformulas for subpart B (formula XXIII or XIV), which can then be coupledto a polymer containing a boronic acid corresponding to any one offormula XXXIII, XXXIV, or XXXV.

The nanoparticles described herein can further include a compound. Insome embodiments the compound can be one or more therapeutic agents,such as a small molecule chemotherapeutic agent or a polynucleotide. Insome embodiments the polynucleotide can be any one or more of DNA, RNA,or interfering RNA (such as shRNA, siRNA or miRNA). In some embodimentsthe small molecule chemotherapeutic agent can be one or more ofcamptothecin, an epothilone, or a taxane. The nanoparticles describedherein can also include a combination of one or more polynucleotideswith one or more small molecule chemotherapeutic agents. In this regard,any one of the polymer of subpart A (formula VI, VII, or VIII) can becombined with any one of the polymer of subpart B (formula XXIII or XIV)to form the polymer containing a polyol nanoparticle segment of thedescribed nanoparticles, the resulting polymer containing a polyol canthen be coupled to a polymer containing a nitrophenylboronic acid andthe polymer of subpart A, subpart B, or the polymer havingnitrophenylboronic acid can be formed with one or more therapeuticagents, such as a small molecule chemotherapeutic agent or apolynucleotide. In some embodiments the polymer of subpart A (formulaVI, VII, or VIII) can be combined with any one of the polymer of subpartB (formula XXIII or XIV) to form the polymer containing a polyolnanoparticle segment of the described nanoparticles, the resultingpolymer containing a polyol can then be coupled to a polymer containinga nitrophenylboronic acid and the polymer of subpart A, subpart B, orthe polymer having nitrophenylboronic acid can be formed with one ormore of DNA, RNA, or interfering RNA (such as shRNA, siRNA or miRNA). Insome embodiments the polymer of subpart A (formula VI, VII, or VIII) canbe combined with any one of the polymer of subpart B (formula XXIII orXIV) to form the polymer containing a polyol nanoparticle segment of thedescribed nanoparticles, the resulting polymer containing a polyol canthen be coupled to a polymer containing a nitrophenylboronic acid andthe polymer of subpart A, subpart B, or the polymer havingnitrophenylboronic acid can be formed with one or more chemotherapeuticagents. In some embodiments the polymer of subpart A (formula VI, VII,or VIII) can be combined with any one of the polymer of subpart B(formula XXIII or XIV) to form the polymer containing a polyolnanoparticle segment of the described nanoparticles, the resultingpolymer containing a polyol can then be coupled to a polymer containinga nitrophenylboronic acid and the polymer of subpart A, subpart B, orthe polymer having nitrophenylboronic acid can be formed with one ormore of DNA, RNA, or interfering RNA (such as shRNA, siRNA or miRNA). Insome embodiments the conjugation between the described polymercontaining a polyol and the described polymer containing anitrophenylboronic acid will be mediated by at least one hydroxyl groupof the nitrophenylboronic acid group. In some embodiments describedherein, the targeted nanoparticles incorporating a therapeutic agent orpolynucleotide can have a polymer containing a polyol formed from thecombination of anyone of the formulas for subpart A (IX, X, or XI) withany one of the formulas for subpart B (formula XXIII or XIV), which canthen be coupled to a polymer containing a nitrophenylboronic acidcorresponding to any one of formula XXXIII, XXXIV, or XXXV.

Targeted Nanoparticles

Described herein are targeted nanoparticles having a polymer containinga polyol that is conjugated to any of 5, 4, 3, 2, or 1 targetingligands. The polymer containing a polyol nanoparticle segment of thetargeted nanoparticles described can have one or more of any one of thefollowing structural units:

where A is an organic moiety of formula

in which R₁ and R₂ are independently selected from any carbon-based ororganic group with a molecular weight of about 10 kDa or less; X isindependently selected from an aliphatic group containing one or more of—H, —F, —C, —N or —O; and Y is independently selected from —OH or anorganic moiety presenting an —OH, and B is an organic moiety linking oneof the R₁ and R₂ of a first moiety A with one of the R₁ and R₂ of asecond moiety A in the polymer. In some embodiments X can beC_(n)H_(2n+1), in which n is any single number from 0-5 and Y is —OH. Insome embodiments A can be any one of:

where the spacer is independently selected from any organic group; theamino acid is selected from any organic group bearing a free amine and afree carboxylic acid group; n is any single number from 1 to 20; and Z₁is independently selected from —NH₂, —OH, —SH, and —COOH; R₁ and R₂independently can have the formula:

wherein d is any single number from 0 to 100, e is any single numberfrom 0 to 100, f is any single number from 0 to 100, Z is a covalentbond linking one organic moiety to another, and Z₁ is independentlyselected from —NH₂, —OH, —SH, and —COOH; B can be any one of

in which q is any single number from 1-20; p is any single number from20-200; and L is a leaving group, where these B subunits are paired withany one of the A subunits described above. In more particularembodiments, the polymer containing a polyol nanoparticle segment of thetargeted nanoparticles shown in structural unit of formula (I) can be:

the polymer containing a polyol nanoparticle segment of the targetednanoparticles shown in structural unit of formula (II) can be:

the polymer containing a polyol nanoparticle segment of the targetednanoparticles shown in structural unit of formula (III) can be:

in which n is any single number from 1-20. In some embodiments of thedescribed targeted nanoparticle, the polymer containing a polyol is:

In summary, any one of the formulas for subpart A (formula VI, VII, orVIII) can be combined with any one of the formulas for subpart B(formula XXIII or XIV) to form the polymer containing a polyol of thedescribed targeted nanoparticles. In certain aspects described hereinthe targeted nanoparticles can have a polymer containing a polyol formedfrom the combination of anyone of the formulas for subpart A (IX, X, orXI) with any one of the formulas for subpart B (formula XXIII or XIV).

The described targeted nanoparticles can also have a polymer containinga boronic acid, coupled to the polymer containing a polyol with areversible covalent linkage. In some embodiments the nanoparticle willbe configured to present the polymer containing a boronic acid to anenvironment external to the nanoparticle. In still further embodiments,the polymer containing the boronic acid is conjugated to a targetingligand at its terminal end opposite the nanoparticle. In someembodiments the polymer containing a boronic acid comprises at least oneterminal boronic acid group and has the general formula:

where R₃ and R₄ are independently an hydrophilic organic polymer, X₁ isan organic moiety containing one or more of —C, —N, or —B, Y₁ is analkyl group of formula —C_(m)H_(2m)—, in which m is ≧1 or an aromaticgroup, r is any single number from 1-1000, a is any single number from0-3, and b is any single number from 0-3 and functional group 1 andfunctional group 2 may be the same or different and may be independentlyselected from any one of —B(OH)₂, —OCH₃, or —OH. In some embodimentsthese variable subparts of the described polymer containing a boronicacid can be selected from the following: R₃ and R₄ may be (CH₂CH₂O)_(t),where t is any single number from 2 to 2000; X₁ is any one of—NH—C(═O)—, —S—S—, —C(═O)—NH—, —O—C(═O)— or —C(═O)—O— and Y₁ is a phenylgroup. In some embodiments these variable subparts of the describedpolymer containing a boronic acid can be selected from the following: R₃and R₄ may be (CH₂CH₂O)_(t), where t is any single number from 2 to2000; X₁ is any one of —NH—C(═O)—, —S—S—, —C(═O)—NH—, —O—C(═O)— or—C(═O)—O— and Y₁ is a phenyl group, and r can have a value of 1, a canhave a value of 0 and b can have a value of 1. In a particularembodiment the targeted nanoparticle of described herein can include apolymer containing a boronic acid having the following formula:

where s is any single number from 20-300.

In some embodiments the polymer containing a boronic acid has anitrophenylboronic acid. In some embodiments the polymer containing anitrophenylboronic acid comprises a nitrophenylboronic acid group andhas the general formula:

where R₃ and R₄ are independently an hydrophilic organic polymer, X₁ isan organic moiety containing one or more of —C, —N, or —B, Y₁ is analkyl group of formula —C_(m)H_(2m)—, in which m is ≧1 or an aromaticgroup, r is any single number from 1-1000, a is any single number from0-3, and b is any single number from 0-3 and functional group 1 andfunctional group 2 may be the same or different and may be independentlyselected from any one of —B(OH)₂, —OCH₃, or —OH. In some embodimentsthese variable subparts of the described polymer containing a boronicacid can be selected from the following: R₃ and R₄ may be (CH₂CH₂O)_(t),where t is any single number from 2 to 2000; X₁ is any one of—NH—C(═O)—, —S—S—, —C(═O)—NH—, —O—C(═O)— or —C(═O)—O— and Y₁ is anitrophenyl group. In some embodiments these variable subparts of thedescribed polymer containing a nitrophenylboronic acid can be selectedfrom the following: R₃ and R₄ may be (CH₂CH₂O)_(t), where t is anysingle number from 2 to 2000; X₁ is any one of —NH—C(═O)—, —S—S—,—C(═O)—NH—, —O—C(═O)— or —C(═O)—O— and Y₁ is a nitrophenyl group, and rcan have a value of 1, a can have a value of 0 and b can have a valueof 1. In each of the embodiments of the polymer containing anitrophenylboronic acid, the nitro group can be at either the ortho,meta, or para position, relative to the boronic acid group, of thephenyl ring. In still further embodiments, the polymer containing anitrophenylboronic acid can have additional groups present on the phenylring, such as a methyl group. In a particular embodiment the targetednanoparticle of described herein can include a polymer containing aboronic acid having any one of the following formulas:

where s is any single number from 20-300. The polymers of formulasXXXIII, XXXIV, and XXXV can be further modified to change the positionof the PEG on the phenyl ring to be in the ortho, meta, or para positionrelative to the boronic acid group.

In summary, any one of the formulas for subpart A (formula VI, VII, orVIII) can be combined with any one of the formulas for subpart B(formula XXIII or XIV) to form the polymer containing a polyolnanoparticle segment of the described targeted nanoparticles, theresulting polymer containing a polyol can then be coupled to a polymercontaining a boronic acid, where the polymer containing a boronic acidis either a phenylboronic acid or a nitrophenylboronic acid. In someembodiments the conjugation between the described polymer containing apolyol and the described polymer containing a boronic acid will bemediated by at least one hydroxyl group of the boronic acid group. Incertain aspects described herein the targeted nanoparticles can have apolymer containing a polyol formed from the combination of anyone of theformulas for subpart A (IX, X, or XI) with any one of the formulas forsubpart B (formula XXIII or XIV), which can then be coupled to a polymercontaining a boronic acid having formula XXX. In some embodimentsdescribed herein, the targeted nanoparticles can have a polymercontaining a polyol formed from the combination of anyone of theformulas for subpart A (IX, X, or XI) with any one of the formulas forsubpart B (formula XXIII or XIV), which can then be coupled to a polymercontaining a boronic acid corresponding to any one of formula XXXI,XXXIII, XXXIV, or XXXV.

In some aspects described herein the nanoparticles formed from eitherthe polymer containing a polyol nanoparticle segment described herein orthe combination of a polymer containing a polyol nanoparticle segmentand a polymer containing a boronic acid are conjugated to a targetingligand to form a targeted nanoparticle having a targeting ligand tonanoparticle ratio of 3:1. In some aspects described herein thenanoparticles formed from either the polymer containing a polyolnanoparticle segment described herein or the combination of a polymercontaining a polyol nanoparticle segment and a polymer containing aboronic acid are conjugated to a targeting ligand to form a targetednanoparticle having a targeting ligand to nanoparticle ratio of 1:1. Insome aspects described herein the nanoparticles formed from either thepolymer containing a polyol nanoparticle segment described herein or thecombination of a polymer containing a polyol nanoparticle segment and apolymer containing a boronic acid are conjugated to one single targetingligand to form a targeted nanoparticle. In some aspects, the describedtargeting ligand is conjugated to the polymer containing a boronic acidat the terminal end opposite the boronic acid. The targeting ligandconjugated to the described targeted nanoparticle can be any one of aprotein, protein fragment, an amino acid peptide, or an aptamer fromeither amino acids or polynucleotides, or other high affinity moleculesknown to bind a target of interest. In some embodiments a targetingligand that is a protein, or protein fragment, can be any one of anantibody, a cellular receptor, a ligand for a cellular receptor, such astransferrin, or a protein or chimeric protein having a portion thereof.Where the targeted ligand is an antibody, the antibody can be a human,murine, rabbit, non-human primate, canine, or rodent antibody, or achimeric composed of any two such antibodies. Furthermore, the antibodymay be humanized such that only the CDR segments or a small portion ofthe variable region comprising a CDR segment is non-human and theremainder of the antibody is human. The antibodies described herein canbe of any isotype, such as IgG, IgM, IgA, IgD, IgE, IgY or another typeof isotype understood to be produced by a mammal. In some embodimentsthe targeting ligand may only include the amino acid peptide from anantibody, a cellular receptor, a ligand for a cellular receptor that isresponsible for binding to its target.

In some aspects, a nanoparticle formed from any one of the formulas forsubpart A (formula VI, VII, or VIII) combined with any one of theformulas for subpart B (formula XXIII or XIV) to form the polymercontaining a polyol nanoparticle segment of the described targetednanoparticles, further coupled to any of 5, 4, 3, 2, or 1 polymerscontaining a boronic acid, such as phenylboronic acid or anitrophenylboronic acid that is conjugated to a targeting ligand.

In some aspects, a nanoparticle formed from any one of the formulas forsubpart A (formula VI, VII, or VIII) combined with any one of theformulas for subpart B (formula XXIII or XIV) to form the polymercontaining a polyol nanoparticle segment of the described targetednanoparticles, further coupled to a single polymer containing a boronicacid, such as phenylboronic acid or a nitrophenylboronic acid that isconjugated to a targeting ligand.

In certain aspects described herein the targeted nanoparticles can havea polymer containing a polyol formed from the combination of anyone ofthe formulas for subpart A (IX, X, or XI) with any one of the formulasfor subpart B (formula XXIII or XIV), which may be coupled to any of 5,4, 3, 2, or 1 polymers containing a boronic acid having formula XXX thatis coupled to a targeting ligand at its terminal end opposite theboronic acid.

In certain aspects described herein the targeted nanoparticles can havea polymer containing a polyol formed from the combination of anyone ofthe formulas for subpart A (IX, X, or XI) with any one of the formulasfor subpart B (formula XXIII or XIV), which may be coupled to a singlepolymer containing a boronic acid having formula XXX that is coupled toa targeting ligand at its terminal end opposite the boronic acid.

In some aspects, a nanoparticle formed from any one of the formulas forsubpart A (formula VI, VII, or VIII) combined with any one of theformulas for subpart B (formula XXIII or XIV) to form the polymercontaining a polyol nanoparticle segment of the described targetednanoparticles, further coupled to any of 5, 4, 3, 2, or 1 polymerscontaining a boronic acid, such as phenylboronic acid or anitrophenylboronic acid that is conjugated to a targeting ligand, wherethe resulting targeted nanoparticle is conjugated to only a singletargeting ligand.

In some aspects, a nanoparticle formed from any one of the formulas forsubpart A (formula VI, VII, or VIII) combined with any one of theformulas for subpart B (formula XXIII or XIV) to form the polymercontaining a polyol nanoparticle segment of the described targetednanoparticles, further coupled to a single polymer containing a boronicacid, such as phenylboronic acid or a nitrophenylboronic acid that isconjugated to a targeting ligand, where the resulting targetednanoparticle is conjugated to only a single targeting ligand.

In certain aspects described herein the targeted nanoparticles can havea polymer containing a polyol formed from the combination of anyone ofthe formulas for subpart A (IX, X, or XI) with any one of the formulasfor subpart B (formula XXIII or XIV), which may be coupled to any of 5,4, 3, 2, or 1 polymers containing a boronic acid having formula XXX thatis coupled to a targeting ligand at its terminal end opposite theboronic acid, where the resulting targeted nanoparticle is conjugated toonly a single targeting ligand.

In certain aspects described herein the targeted nanoparticles can havea polymer containing a polyol formed from the combination of anyone ofthe formulas for subpart A (IX, X, or XI) with any one of the formulasfor subpart B (formula XXIII or XIV), which may be coupled to a singlepolymer containing a boronic acid having formula XXX that is coupled toa targeting ligand at its terminal end opposite the boronic acid, wherethe resulting targeted nanoparticle is conjugated to only a singletargeting ligand.

In some embodiments the targeted nanoparticles described herein areconjugated to any a targeting ligand. In some embodiments the targetednanoparticles described herein are conjugated to a single targetingligand. In some embodiments described herein, the targeted nanoparticlescan have a polymer containing a polyol formed from the combination ofanyone of the formulas for subpart A (IX, X, or XI) with any one of theformulas for subpart B (formula XXIII or XIV), which can then be coupledto any of 5, 4, 3, 2, or 1 polymers containing a boronic acidcorresponding to any one of formula XXXI, XXXIII, XXXIV, or XXXV, thatis further conjugated to a targeting ligand selected from one or more ofa protein, protein fragment, an amino acid peptide, or an aptamer.

In some embodiments the targeted nanoparticles described herein areconjugated to a single targeting ligand. In some embodiments describedherein, the targeted nanoparticles can have a polymer containing apolyol formed from the combination of anyone of the formulas for subpartA (IX, X, or XI) with any one of the formulas for subpart B (formulaXXIII or XIV), which can then be coupled to a polymer containing aboronic acid corresponding to any one of formula XXXI, XXXIII, XXXIV, orXXXV, that is further conjugated to a single targeting ligand selectedfrom a protein, protein fragment, an amino acid peptide, or an aptamer.

In some embodiments the targeted nanoparticles described herein areconjugated to a single targeting ligand. In some embodiments describedherein, the targeted nanoparticles can have a polymer containing apolyol formed from the combination of anyone of the formulas for subpartA (IX, X, or XI) with any one of the formulas for subpart B (formulaXXIII or XIV), which can then be coupled to any of 5, 4, 3, 2, or 1polymers containing a boronic acid corresponding to any one of formulaXXXI, XXXIII, XXXIV, or XXXV, that is further conjugated to an antibody,cellular receptor, ligand for a cellular receptor, or a protein orchimeric protein having a portion thereof

In some embodiments described herein, the targeted nanoparticles canhave a polymer containing a polyol formed from the combination of anyoneof the formulas for subpart A (IX, X, or XI) with any one of theformulas for subpart B (formula XXIII or XIV), which can then be coupledto a polymer containing a boronic acid corresponding to any one offormula XXXI, XXXIII, XXXIV, or XXXV, that is further conjugated to asingle antibody, cellular receptor, ligand for a cellular receptor, or aprotein or chimeric protein having a portion thereof.

In some embodiments the targeted nanoparticles described herein areconjugated to a single targeting ligand. In some embodiments describedherein, the targeted nanoparticles can have a polymer containing apolyol formed from the combination of anyone of the formulas for subpartA (IX, X, or XI) with any one of the formulas for subpart B (formulaXXIII or XIV), which can then be coupled to a polymer containing aboronic acid corresponding to any one of formula XXXI, XXXIII, XXXIV, orXXXV, that is further conjugated to a single targeting ligand selectedfrom a protein, protein fragment, an amino acid peptide, or an aptamer,where the resulting targeted nanoparticle is conjugated to only a singletargeting ligand.

In some embodiments the targeted nanoparticles described herein areconjugated to a single targeting ligand. In some embodiments describedherein, the targeted nanoparticles can have a polymer containing apolyol formed from the combination of anyone of the formulas for subpartA (IX, X, or XI) with any one of the formulas for subpart B (formulaXXIII or XIV), which can then be coupled to any of 5, 4, 3, 2, or 1polymers containing a boronic acid corresponding to any one of formulaXXXI, XXXIII, XXXIV, or XXXV, that is further conjugated to an antibody,cellular receptor, ligand for a cellular receptor, or a protein orchimeric protein having a portion thereof, where the resulting targetednanoparticle is conjugated to only a single targeting ligand.

In some embodiments described herein, the targeted nanoparticles canhave a polymer containing a polyol formed from the combination of anyoneof the formulas for subpart A (IX, X, or XI) with any one of theformulas for subpart B (formula XXIII or XIV), which can then be coupledto a polymer containing a boronic acid corresponding to any one offormula XXXI, XXXIII, XXXIV, or XXXV, that is further conjugated to asingle antibody, cellular receptor, ligand for a cellular receptor, or aprotein or chimeric protein having a portion thereof, where theresulting targeted nanoparticle is conjugated to only a single targetingligand.

The targeted nanoparticles described herein can further include acompound. In some embodiments the compound can be one or moretherapeutic agents, such as a small molecule chemotherapeutic agent or apolynucleotide. In some embodiments the polynucleotide can be any one ormore of DNA, RNA, or interfering RNA (such as shRNA, siRNA or miRNA). Insome embodiments the small molecule chemotherapeutic agent can be one ormore of camptothecin, an epothilone, or a taxane. The targetednanoparticles described herein can also include a combination of one ormore polynucleotides with one or more small molecule chemotherapeuticagents.

Having discussed the various types of nanoparticles and targetednanoparticles that can be produced using the components describedherein, the following particular embodiments can be produced. In oneembodiment the described targeted nanoparticle has a mucicacid-containing polymer, a therapeutic agent selected from camptothecin,an epothilone, a taxane, or an interfering RNA sequence, a polymercontaining a phenylboronic acid, having formula XXXI, XXXIII, XXXIV, orXXXV, that is coupled to the mucic acid polymer with a reversiblecovalent linkage, and the targeted nanoparticle is configured to presentthe polymer containing the phenylboronic acid to an environment externalto the nanoparticle, where the polymer containing the phenylboronic acidis conjugated to a targeting ligand at its terminal end opposite thenanoparticle, wherein the targeted nanoparticle comprises one singletargeting ligand.

In one embodiment the described targeted nanoparticle has a mucicacid-containing polymer, a therapeutic agent selected from camptothecin,an epothilone, a taxane, or an interfering RNA sequence, a polymercontaining a phenylboronic acid, having formula XXXI, XXXIII, XXXIV, orXXXV, that is coupled to the mucic acid polymer with a reversiblecovalent linkage, and the targeted nanoparticle is configured to presentthe polymer containing the phenylboronic acid to an environment externalto the nanoparticle, where the polymer containing the phenylboronic acidis conjugated to an antibody at its terminal end opposite thenanoparticle, wherein the targeted nanoparticle comprises one singleantibody.

In one embodiment the described targeted nanoparticle has a mucicacid-containing polymer, a therapeutic agent selected from camptothecin,an epothilone, a taxane, or an interfering RNA sequence, a polymercontaining a phenylboronic acid, having formula XXXI, XXXIII, XXXIV, orXXXV, that is coupled to the mucic acid polymer with a reversiblecovalent linkage, and the targeted nanoparticle is configured to presentthe polymer containing the phenylboronic acid to an environment externalto the nanoparticle, where the polymer containing the phenylboronic acidis conjugated to any one of a human, murine, rabbit, non-human primate,canine, or rodent antibody, or a chimeric antibody composed of any twosuch antibodies, where the antibody is any one of an IgG, IgD, IgM, IgE,IgA or IgY isotype, at its terminal end opposite the nanoparticle,wherein the targeted nanoparticle comprises one single antibody.

In one embodiment the described targeted nanoparticle has a mucicacid-containing polymer, a therapeutic agent selected from camptothecin,an epothilone, a taxane, or an interfering RNA sequence, a polymercontaining a phenylboronic acid, having formula XXXI, XXXIII, XXXIV, orXXXV, that is coupled to the mucic acid polymer with a reversiblecovalent linkage, and the targeted nanoparticle is configured to presentthe polymer containing the phenylboronic acid to an environment externalto the nanoparticle, where the polymer containing the phenylboronic acidis conjugated to a cellular receptor at its terminal end opposite thenanoparticle, wherein the targeted nanoparticle comprises one singlecellular receptor.

In one embodiment the described targeted nanoparticle has a mucicacid-containing polymer, a therapeutic agent selected from camptothecin,an epothilone, a taxane, or an interfering RNA sequence, a polymercontaining a phenylboronic acid, having formula XXXI, XXXIII, XXXIV, orXXXV, that is coupled to the mucic acid polymer with a reversiblecovalent linkage, and the targeted nanoparticle is configured to presentthe polymer containing the phenylboronic acid to an environment externalto the nanoparticle, where the polymer containing the phenylboronic acidis conjugated to a receptor ligand at its terminal end opposite thenanoparticle, wherein the targeted nanoparticle comprises one singlereceptor ligand.

In view of the forgoing description, the following items are provided toillustrate particular embodiments of the described subject matter:

-   -   1. A targeted nanoparticle comprising a polymer containing a        polyol, wherein the nanoparticle is conjugated to one single        targeting ligand.    -   2. The targeted nanoparticle of item 1, wherein the targeting        ligand comprises a protein, protein fragment, or amino acid        peptide.    -   3. The targeted nanoparticle of item 2, wherein the protein        comprises an antibody, transferrin, a ligand for a cellular        receptor, or a cellular receptor protein, wherein the targeting        ligand may alternatively comprise a fragment of an antibody,        transferrin, a ligand for a cellular receptor, an aptamer, or a        cellular receptor protein.    -   4. The targeted nanoparticle of item 2 or 3, wherein the protein        fragment comprises an antibody fragment.    -   5. The targeted nanoparticle of item 2 or 3, wherein the amino        acid peptide comprises an amino acid sequence from an antibody.    -   6. The targeted nanoparticle of any one previous item further        comprising a therapeutic agent.    -   7. The targeted nanoparticle of item 6, wherein the therapeutic        agent is a small molecule chemotherapeutic agent or a        polynucleotide.    -   8. The targeted nanoparticle of item 7, wherein the        polynucleotide is interfering RNA.    -   9. The targeted nanoparticle of any one of items 6 to 8, wherein        the targeting ligand is transferrin, or an antibody and the        nanoparticle further comprises a therapeutic agent selected from        camptothecin, an epothilone, a taxane and a polynucleotide or        any combination thereof    -   10. A method of delivering a nanoparticle to a target in a        subject, comprising obtaining a nanoparticle conjugated to one        single targeting ligand that specifically binds to said target        and administering said nanoparticle conjugated to one single        targeting ligand to the subject.    -   11. The method of item 10, wherein the targeting ligand        comprises a protein, protein fragment, or amino acid peptide.    -   12. The method of item 11, wherein the protein comprises an        antibody, transferrin, a ligand for a cellular receptor, an        aptamer, or a cellular receptor protein, wherein the targeting        ligand may alternatively comprise a fragment of an antibody,        transferrin, a ligand for a cellular receptor, or a cellular        receptor protein.    -   13. The method of item 11 or 12, wherein the protein fragment        comprises an antibody fragment.    -   14. The method of item 11 or 12 wherein the amino acid peptide        comprises an amino acid sequence from an antibody.    -   15. The method of any one of items 10-14 wherein the        nanoparticle conjugated to one single targeting ligand further        comprises a therapeutic agent.    -   16. The method of item 15, wherein the therapeutic agent is a        small molecule chemotherapeutic agent or a polynucleotide.    -   17. The method of item 16, wherein the polynucleotide is        interfering RNA.    -   18. The method of item 15, 16 or 17 wherein the targeting ligand        is transferrin, or an antibody and the nanoparticle further        comprises a therapeutic agent selected from camptothecin, an        epothilone, a taxane and a polynucleotide or any combination        thereof    -   19. A targeted nanoparticle comprising a polymer containing a        polyol and a polymer containing a boronic acid, said boronic        acid being coupled to the polymer containing a polyol with a        reversible covalent linkage, said nanoparticle being configured        to present the polymer containing a boronic acid to an        environment external to the nanoparticle, wherein the polymer        containing the boronic acid is conjugated to a targeting ligand        at its terminal end opposite the nanoparticle.    -   20. The targeted nanoparticle of item 19, wherein the polymer        containing a polyol comprises one or more of at least one of the        following structural units

wherein

A is an organic moiety of formula

in which

-   -   R₁ and R₂ are independently selected from any carbon-based or        organic group with a molecular weight of about 10 kDa or less;    -   X is independently selected from an aliphatic group containing        one or more of —H, —F, —C, —N or —O; and    -   Y is independently selected from —OH or an organic moiety        presenting an —OH,        and

B is an organic moiety linking one of the R₁ and R₂ of a first saidmoiety A with one of the R₁ and R₂ of a second said moiety A in thepolymer.

-   -   21. The targeted nanoparticle of item 20, wherein R₁ and R₂        independently have the formula:

wherein

d is from 0 to 100;

e is from 0 to 100;

f is from 0 to 100;

Z is a covalent bond linking one organic moiety to another, and

Z₁ is independently selected from —NH₂, —OH, —SH, and —COOH.

-   -   22. The targeted nanoparticle of item 20 or 21,        wherein

X is C_(n)H_(2n+1), in which n is to 0-5; and

wherein

Y is —OH.

-   -   23. The targeted nanoparticle of item 20, wherein A is        independently selected from the group consisting of

wherein

the spacer is independently selected from any organic group;

the amino acid is selected from any organic group bearing a free amineand a free carboxylic acid group;

n is 1-20; and

Z₁ is independently selected from —NH₂, —OH, —SH, and —COOH.

-   -   24. The targeted nanoparticle of item 23, wherein A is        independently selected from

-   -   25. The targeted nanoparticle of any one of items 20-24, wherein        B is

in which

q is 1-20;

p is 20-200; and

L is a leaving group.

-   -   26. The targeted nanoparticle of item 20, wherein the structural        unit of formula (I) is:

-   -   27. The targeted nanoparticle of item 20, wherein the structural        unit of formula (II) is:

-   -   28. The targeted nanoparticle of item 20, wherein the structural        unit of formula (III) is:

in which

n is 1-20.

-   -   29. The targeted nanoparticle of item 19, wherein the polymer        containing a polyol is

-   -   30. The targeted nanoparticle of any one of items 19 to 29,        wherein the polymer containing a boronic acid comprises at least        one terminal boronic acid group and has the general formula:

wherein

R₃ and R₄ are independently an hydrophilic organic polymer,

X₁ is an organic moiety containing one or more of —C, —N, or —B,

Y₁ is an alkyl group of formula —C_(m)H_(2m)—, in which m is ≧1 or anaromatic group,

r is 1-1000,

a is 0-3, and

b is 0-3.

-   -   31. The targeted nanoparticle of item 30, wherein R₃ and R₄ are        (CH₂CH₂O)₁, where t is from 2 to 2000.    -   32. The targeted nanoparticle of item 30 or 31, wherein X₁ is        —NH—C(═O)—, —S—S—, —C(═O)—NH—, —O—C(═O)— or —C(═O)—O— and Y₁ is        a phenyl group.    -   33. The targeted nanoparticle of any one of items 30-32, wherein        r=1, a=0 and b=1.

34. The targeted nanoparticle of any one of items 30-33, whereinfunctional group 1 and functional group 2 are the same or different andare independently selected from —B(OH)₂, —OCH₃, —OH.

-   -   35. The targeted nanoparticle of any one of items 19-34, wherein        the polymer containing a boronic acid is:

wherein s=20-300.

-   -   36. The targeted nanoparticle of any one of items 19-35, wherein        the polymer containing a boronic acid is conjugated to a        targeting ligand at the terminal end opposite the boronic acid.    -   37. The targeted nanoparticle of any one of items 19-36, wherein        the targeting ligand is any one of an antibody, transferrin, a        ligand for a cellular receptor, an aptamer, or a cellular        receptor protein, wherein the targeting ligand may alternatively        comprise a fragment of an antibody, transferrin, a ligand for a        cellular receptor, or a cellular receptor protein.    -   38. The targeted nanoparticle of any one of items 19-37 further        comprising a therapeutic agent.    -   39. The targeted nanoparticle of item 38, wherein the        therapeutic agent is a small molecule chemotherapeutic agent or        a polynucleotide.    -   40. The targeted nanoparticle of item 39, wherein the        polynucleotide is interfering RNA.    -   41. The targeted nanoparticle of item 39, wherein the small        molecule chemotherapeutic agent is selected from camptothecin,        an epothilone, a taxane and a polynucleotide or any combination        thereof    -   42. The targeted nanoparticle of any one of items 19-29, wherein        the polymer containing a boronic acid has a nitrophenylboronic        acid group and has the general formula:

wherein

R₃ and R₄ are independently an hydrophilic organic polymer,

X₁ is an organic moiety containing one or more of —C, —N, or —B,

Y₁ is a phenyl group having a nitro group,

r is 1-1000,

a is 0-3, and

b is 0-3.

-   -   43. The targeted nanoparticle of item 42, wherein R₃ and R₄ are        (CH₂CH₂O)_(t), where t is from 2 to 2000.    -   44. The targeted nanoparticle of any one of items 42-43, wherein        X₁ is —NH—C(═O)—, —S—S—, —C(═O)—NH—, —O—C(═O)— or —C(═O)—O— and        Y₁ is a phenyl group having a nitro group.    -   45. The targeted nanoparticle of any one of items 42-44, wherein        r=1, a=0 and b=1.    -   46. The targeted nanoparticle of any one of items 42-45, wherein        functional group 1 and functional group 2 are the same or        different and are independently selected from —B(OH)₂, —OCH₃,        —OH.    -   47. The targeted nanoparticle of any one of items 19-29 or        42-46, wherein the polymer containing a nitrophenylboronic acid        any one of:

wherein s=20-300.

-   -   48. The targeted nanoparticle of any one of items 42-47, wherein        the polymer containing a nitrophenylboronic acid is conjugated        to a targeting ligand at the terminal end opposite the        nitrophenylboronic acid.    -   49. The targeted nanoparticle of any one of items 42-48, wherein        the targeting ligand is any one of an antibody, transferrin, a        ligand for a cellular receptor, an aptamer, or a cellular        receptor protein, wherein the targeting ligand may alternatively        comprise a fragment of an antibody, transferrin, a ligand for a        cellular receptor, or a cellular receptor protein.    -   50. The targeted nanoparticle of any one of items 42-49 further        comprising a therapeutic agent.    -   51. The nanoparticle of item 50, wherein the therapeutic agent        is a small molecule chemotherapeutic agent or a polynucleotide.    -   52. The targeted nanoparticle of item 51, wherein the        polynucleotide is interfering RNA.    -   53. The targeted nanoparticle of item 51, wherein the small        molecule chemotherapeutic agent is selected from camptothecin,        an epothilone, a taxane and a polynucleotide or any combination        thereof    -   54. A method of delivering a nanoparticle to a target in a        subject, comprising obtaining a targeted nanoparticle of any one        of items 19-53, wherein the nanoparticle is conjugated to one        single targeting ligand that specifically binds to said target        and administering the nanoparticle conjugated to one single        targeting ligand to the subject.    -   55. The method of item 54, wherein the targeting ligand is any        one of an antibody, transferrin, a ligand for a cellular        receptor, an aptamer or a cellular receptor protein, wherein the        targeting ligand may alternatively comprise a fragment of an        antibody, transferrin, a ligand for a cellular receptor, or a        cellular receptor protein.    -   56. The method of item 55, wherein the targeted nanoparticle        further comprises a therapeutic agent.    -   57. The method of item 56, wherein the therapeutic agent is a        small molecule chemotherapeutic agent or a polynucleotide.    -   58. The method of item 57, wherein the polynucleotide is        interfering RNA.    -   59. The method of item 57, wherein the small molecule        chemotherapeutic agent is selected from camptothecin, an        epothilone, a taxane and a polynucleotide or any combination        thereof    -   60. A method of delivering a nanoparticle to a target in a        subject, comprising obtaining a nanoparticle of any one of items        19-59, wherein the nanoparticle is conjugated to one single        targeting ligand that specifically binds to said target and        administering the nanoparticle conjugated to one single        targeting ligand to the subject.    -   61. The method of item 60, wherein the targeting ligand is any        one of an antibody, transferrin, a ligand for a cellular        receptor, an aptamer, or a cellular receptor protein, wherein        the targeting ligand may alternatively comprise a fragment of an        antibody, transferrin, a ligand for a cellular receptor, or a        cellular receptor protein.    -   62. The method of item 61 wherein the targeted nanoparticle        further comprises a therapeutic agent.    -   63. The method of item 62, wherein the therapeutic agent is a        small molecule chemotherapeutic agent or a polynucleotide.    -   64. The method of item 63, wherein the polynucleotide is        interfering RNA.    -   65. The method of item 63, wherein the small molecule        chemotherapeutic agent is selected from camptothecin, an        epothilone, a taxane and a polynucleotide or any combination        thereof    -   66. A targeted nanoparticle comprising a mucic acid-containing        polymer, a therapeutic agent, a polymer containing a        phenylboronic acid, said phenylboronic acid being coupled to the        mucic acid polymer with a reversible covalent linkage, said        targeted nanoparticle being configured to present the polymer        containing the phenylboronic acid to an environment external to        the nanoparticle, wherein the polymer containing the        phenylboronic acid is conjugated to a targeting ligand at its        terminal end opposite the nanoparticle, wherein said targeted        nanoparticle comprises one single targeting ligand.    -   67. The targeted nanoparticle of item 66 wherein the therapeutic        agent is any one of camptothecin, an epothilone, a taxane, a        polynucleotide or any combination thereof; the polymer        containing phenylboronic acid any one of:

wherein s=20-300, and the targeting ligand comprises any one of anantibody, transferrin, a ligand for a cellular receptor, or a cellularreceptor protein, wherein the targeting ligand may alternativelycomprise a fragment of an antibody, transferrin, a ligand for a cellularreceptor, or a cellular receptor protein.

-   -   68. The targeted nanoparticle of item 66 wherein the therapeutic        agent is camptothecin, the polymer containing phenylboronic acid        any one of:

wherein s=20-300, and the targeting ligand comprises an antibody.

-   -   69. A method of producing a targeted nanoparticle of any one of        items 1-9, 19-53, or 66-68, comprising conjugating a        nanoparticle comprising a polymer containing a polyol with a        polymer containing a boronic acid and conjugating the polymer        containing a boronic acid to a targeting ligand, wherein the        targeted nanoparticle has a ratio of the nanoparticle to the        targeting ligand of no more than 3 to 1, or no more than 2 to 1,        or no more than 1 to 1.    -   70. The method of item 69, wherein the targeted nanoparticle has        a ratio of the nanoparticle to the targeting ligand of 1 to 1.    -   71. A method of item 69 or 70, wherein the polymer containing a        polyol is a mucic acid polymer, the polymer containing a boronic        acid comprises a phenylboronic acid or nitrophenylboronic acid,        and the targeting ligand comprises an antibody, transferrin, a        ligand for a cellular receptor, an aptamer, or a cellular        receptor protein.    -   72. A method of any one of items 69-71, wherein the polymer        containing phenylboronic acid or nitrophenylboronic acid is one        of the following compounds:

wherein s=20-300.

-   -   73. The method of any one of items 69-72, wherein the polymer        containing a boronic acid is conjugated to the targeting ligand        before it is conjugated to the nanoparticle comprising a polymer        containing a polyol.    -   74. A kit for producing a targeted nanoparticle of any one of        items 1-9, 19-53, or 66-68 comprising a nanoparticle comprising        a polymer containing a polyol, a polymer containing a boronic        acid, a targeting ligand, and instructions for producing a        targeted nanoparticle having a nanoparticle to targeting ligand        ratio of no more than 3 to 1, or no more than 2 to 1, or no more        than 1 to 1.    -   75. The kit of item 74, wherein instructions describe how to        produce a targeted nanoparticle with a nanoparticle to targeting        ligand ratio of 1 to 1.    -   76. The kit of item 74 or 75, wherein the polymer containing a        polyol is a mucic acid polymer, the polymer containing a boronic        acid comprises a phenylboronic acid or nitrophenylboronic acid,        and the targeting ligand comprises an antibody, transferrin, a        ligand for a cellular receptor, or a cellular receptor protein.    -   77. The kit of any one of items 74-76, wherein the polymer        containing phenylboronic acid or nitrophenylboronic acid is one        of the following compounds:

wherein s=20-300.

-   -   78. The kit of any one of items 74-77, wherein the polymer        containing a boronic acid and the targeting ligand are provided        as a single conjugated unit.    -   79. A kit of any one of items 74-78, wherein the kit further        comprises one or more reagents for conjugating a targeting        ligand to the polymer containing a boronic acid.    -   80. A method of treating a subject having a disease condition,        comprising obtaining a nanoparticle of any one of items 1-9,        19-37, 40, 42-49, 52, or 66-67 that further comprises a        therapeutic agent for treating said disease condition, wherein        the polymer containing boronic acid is conjugated to one single        targeting ligand that specifically binds to cells contributing        to said disease condition and administering said nanoparticle to        the subject.    -   81. The method of item 80, wherein the disease condition is        cancer and the therapeutic agent is a chemotherapeutic compound.    -   82. The method of item 81, wherein the chemotherapeutic compound        is any one of camptothecin, an epothilone, or a taxane.    -   83. A method of treating a subject having a disease condition,        comprising obtaining a nanoparticle of any one of items 1-9,        19-37, 40, 42-49, 52, or 66-67 that further comprises a        therapeutic agent for treating said disease condition, wherein        the polymer containing a nitrophenylboronic acid is conjugated        to one single targeting ligand that specifically binds to cells        contributing to said disease condition and administering said        nanoparticle to the subject.    -   84. The method of item 83, wherein the disease condition is        cancer and the therapeutic agent is a chemotherapeutic compound.    -   85. The method of item 84, wherein the chemotherapeutic compound        is any one of camptothecin, an epothilone, or a taxane.    -   86. The method of any one of items 69-72, wherein the polymer        containing a boronic acid is conjugated to the targeting ligand        after it is conjugated to the nanoparticle comprising a polymer        containing a polyol.    -   87. A composition comprising the nanoparticle of any one of        items 1-9, 19-53, or 66-68, and a suitable vehicle and/or        excipient.

EXAMPLES

The methods system herein described are further illustrated in thefollowing examples, which are provided by way of illustration and arenot intended to be limiting. A person skilled in the art will appreciatethe applicability of the features described in detail for methods ofnucleic acid detection and detection of other targets, such as proteins,antigens, eukaryotic or prokaryotic cells, and the like.

All chemical reagents were obtained from commercial suppliers and wereused as received without further purification. Polymer samples wereanalyzed on a Viscotek GPC System equipped with a TDA 302 tripledetector array consisting of a differential refractive index (RI)detector, a differential viscometer and a low angle light scatteringdetector. A 7.5% acetic acid solution was used as eluant at a 1 mL/minflow rate.

pGL3, a plasmid containing the firefly luciferase gene was extracted andpurified from bacteria expressing pGL3. siGL3 was purchased fromIntegrated DNA Technologies (sequence provided below). siCON1 (sequenceprovided below) was purchased from Dharmacon. HeLa cells were used todetermine the efficacy of pDNA or siRNA delivery by the cationic mucicacid diamine-DMS polymer.

TABLE 1 siRNA sequences Plas- mid Sequences SEQ ID NO siGL3GUGCCAGAGUCCUUCGAUAdTdT (sense) SEQ ID NO: 1UAUCGAAGGACUCUGGCACdTdT (antisense) SEQ ID NO: 2 siCON1UAGCGACUAAACACAUCAAUU (sense) SEQ ID NO: 3UUGAUGUGUUUAGUCGCUAUU (antisense) SEQ ID NO: 4

Example 1 Synthesis of Mucic Acid Dimethyl Ester, (1)

5 g (22.8 mmol) of mucic acid (Aldrich) was added to a 500 mL roundbottom flask containing 120 mL of methanol and 0.4 mL of concentratedsulfuric acid. This mixture was allowed to reflux at 85° C. overnightunder constant stirring. The mixture was subsequently filtered, washedwith methanol and then recrystallized from a mixture of 80 mL methanoland 0.5 mL triethylamine. After drying under vacuum overnight, 8.0 g(33.6 mmol, 71%) of mucic acid dimethyl ester was obtained. ¹H NMR((CD₃)₂SO) δ 4.88-4.91 (d, 2H), 4.78-4.81 (m, 2H), 4.28-4.31 (d, 2H),3.77-3.78 (d, 2H), 3.63 (s, 6H). ESI/MS (m/z): 261.0 [M+Na]⁺

Example 2 Synthesis of N—BOC-Protected Mucic Acid Diamine, (2)

A mixture of 8 g (33.6 mmol) of Mucic Acid Dimethyl Ester (1; Example1), 12.4 mL (88.6 mmol) triethylamine and 160 mL methanol was heatedunder reflux at 85° C. in a 500 mL round bottom flask under constantstirring for 0.5 h prior to the addition of 14.2 g (88.6 mmol) N—BOCdiamine (Fluka) dissolved in methanol (32 mL). This reaction suspensionwas then returned to reflux. After refluxing overnight, the mixture wasfiltered, washed with methanol, recrystallized from methanol and thendried under vacuum to yield 9.4 g (19 mmol, 57%) of N—BOC-ProtectedMucic Acid Diamine. ¹H NMR ((CD₃)₂SO) δ 7.66 (m, 2H), 6.79 (m, 2H),5.13-5.15 (d, 2H), 4.35-4.38 (d, 2H), 4.08-4.11 (m, 2H), 3.78-3.80 (d,2H), 2.95-3.15 (m, 8H), 1.38 (s, 18). ESI/MS (m/z): 517.1 [M+Na]⁺

Example 3 Synthesis of Mucic Acid Diamine, (3)

8 g (16.2 mmol) of the N—BOC-Protected Mucic Acid Diamine (2; Example 2)was transferred to a 500 mL round bottom flask containing 3 M HCl inmethanol (160 mL) and allowed to reflux overnight at 85° C. underconstant stirring. The precipitate was subsequently filtered, washedwith methanol and vacuum dried overnight to give 5.7 g (15.6 mmol, 96%)of Mucic Acid Diamine. ¹H NMR ((CD₃)₂SO) δ 7.97 (m, 8H), 5.35-5.38 (m,2H), 4.18-4.20 (m, 2H), 3.82 (m, 2H), 3.35-3.42 (m, 8H), 2.82-2.90 (m,4H). ESI/MS (m/z): 294.3 [M]⁺, 317.1 [M+Na]⁺, 333.0 [M+K]⁺

Example 4 Mucic Acid Diamine-DMS Copolymer (MAP), (4)

A 1.5 mL eppendorff tube was charged with a solution of 85.5 mg (0.233mmol) of the bis(hydrochloride) salt of Example 3 (3) in 0.8 mL of 0.1 MNaHCO₃. Dimethylsuberimidate.2HCl (DMS, Pierce Chemical Co., 63.6 mg,0.233 mmol) was added and the solution was vortexed and centrifuged todissolve the components. The resulting mixture was stirred at roomtemperature for 15 h. The mixture was then diluted to 8 mL with waterand the pH was brought to 4 with the addition of 1 N HCl. This solutionwas then dialyzed with a 3500 MWCO dialysis membrane (Pierce pleateddialysis tubing) in ddH₂O for 24 h. The dialyzed solution waslyophilized to dryness to give 49 mg of a white fluffy powder. ¹H NMR(500 MHz, dDMSO) δ 9.15 (bs), 7.92 (bs), 5.43 (bs), 4.58 (bs), 4.17(bs), 3.82 (bs), 3.37 (bs), 3.28 (bs), 2.82 (bs), 2.41 (bs), 1.61 (bs),1.28 (bs). ¹³C NMR (126 MHz, dDMSO) δ 174.88 (s, 1H), 168.38 (s, 1H),71.45 (s, 4H), 71.22 (s, 3H), 42.34 (s, 2H), 36.96 (s, 3H), 32.74 (s,3H), 28.09 (s, 4H), 26.90 (s, 4H). Mw [GPC]=2520, Mw/Mn=1.15.

Polymer 4 is an example of a cationic A-B type (repeating structure isABABAB . . . ) polymer containing a polyol.

Example 5 Boronic Acid-Amide-PEG₅₀₀₀, (5)

When a polymer of Example 4 is assembled with nucleic acids, e.g.,siRNAs, they will form nanoparticles. These nanoparticles will need tohave steric stabilization to be used in mammals and optionally theycould have targeting agents included. To perform these two functions,the nanoparticles can be decorated with PEG for steric stabilization andPEG-targeting ligands. To do so, PEG compounds containing boronic acidsare prepared. For example, a PEG containing boronic acid can besynthesized according the example below.

332 mg of 4-carboxyphenylboronic acid (2 mmol) was dissolved in 8 mL ofSOCl₂. To this was added a few drops of DMF and the mixture was refluxedunder argon for 2 h. Excess SOCl₂ was removed under reduced pressure andthe resulting solid was dissolved in 10 mL of anhydrous dichloromethane.To this solution was added 500 mg of PEG₅₀₀₀-NH₂ (2 mmol) and 418 μL oftriethylamine (60 mmol) dissolved in 5 mL of dichloromethane at 0° C.under argon. The resulting mixture was warmed to room temperature andstirring was continued overnight. The dichloromethane solvent wasremoved under reduced pressure and the resulting liquid was precipitatedwith 20 mL of diethyl ether. The precipitate was filtered, dried andre-dissolved in ddH₂O. The aqueous solution was then filtered with a0.45 μm filter and dialyzed with a 3500 MWCO dialysis membrane (Piercepleated dialysis tubing) in ddH₂O for 24 h. The dialyzed solution waslyophilized to dryness. ¹H NMR (300 MHz, dDMSO) δ 7.92-7.77 (m), 4.44(d), 4.37 (t), 3.49 (m), 2.97 (s).

Example 6 Boronic Acid-Disulfide-PEG₅₀₀₀, (6)

A cleavable version (under reducing conditions) of the PEG compound ofExample 5 can also be synthesized as follows.

250 mg of PEG₅₀₀₀-SH (0.05 mmol, LaySanBio Inc.) was added to a glassvial equipped with a stirbar. To this was added 110 mg of aldrithiol-2(0.5 mmol, Aldrich) dissolved in 4 mL of methanol. The solution wasstirred at room temperature for 2 h after which, 77 mg ofmercaptophenylboronic acid (0.5 mmol, Aldrich) in 1 mL of methanol wasadded. The resulting solution was stirred for an additional 2 h at roomtemperature. Methanol was removed under vacuo and the residue wasre-dissolved in 2 mL of dichloromethane. 18 mL of diethyl ether wasadded to the dichloromethane solution and the mixture was allowed to sitfor 1 h. The resulting precipitate was collected via centrifugation,washed several times with diethyl ether and dried. The dried solid wasre-dissolved in water, filtered with a 0.45 μm filter and dialyzed witha 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) in ddH₂Ofor 15 h. The dialyzed solution was lyophilized to dryness. ¹H NMR (300MHz, dDMSO) δ 8.12-8.00 (m), 7.83-7.72 (m), 7.72-7.61 (m), 7.61-7.43(m), 3.72 (d, J=5.4), 3.68-3.15 (m), 3.01-2.83 (m).

Example 7 Synthesis of (2,3,5,6)-Tetrafluorophenyl Boronic Acid-PEG₅₀₀₀,(7)

A fluorinated version of the PEG compound containing boronic acids ofExample 5 can be synthesized and used as an imaging agent with thetherapeutic nanoparticle. The fluorine atoms for imaging can beincorporated as described and illustrated below.

(2,3,5,6)-fluorocarboxyphenylboronic acid is dissolved in excess SOCl₂(˜100 eq.) and to it is added a few drops of DMF. The mixture isrefluxed under argon for 2 h. Excess SOCl₂ is removed under reducedpressure and the resulting residue is dissolved in anhydrousdichloromethane. To this solution, PEG₅₀₀₀-NH₂ (1 eq.) and triethylamine(30 eq,) dissolved in dichloromethane is added at 0° C. under argon. Theresulting mixture is warmed to room temperature and stirring iscontinued overnight. The dichloromethane solvent is removed underreduced pressure and the resulting liquid is precipitated with diethylether. The precipitate is filtered, dried and re-dissolved in ddH₂O. Theaqueous solution is then filtered with a 0.45 μm filter and dialyzedwith a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) inddH₂O for 24 h. The dialyzed solution is lyophilized to dryness.

The fluorine containing compound is useful to provide ¹⁹F in thenanoparticle. The ¹⁹F can be detected by magnetic resonance spectroscopyusing a standard patient MRI. The addition of the ¹⁹F enables thenanoparticles to be imaged (can be done for just imaging or with theaddition of a therapeutic agent can allow for imaging and therapy).

Example 8 Synthesis of (2,3,5,6)-Tetrafluorophenyl BoronicAcid-Disulfide-PEG₅₀₀₀, (8)

A fluorinated version of the cleavable PEG compound containing boronicacids of Example 5 can be synthesized and used as an imaging agent withthe therapeutic nanoparticle. The fluorine atoms for imaging can beincorporated as described and illustrated below.

250 mg of PEG₅₀₀₀-SH (0.05 mmol, LaySanBio Inc.) are added to a glassvial equipped with a stirbar. To this is added 110 mg of aldrithiol-2(0.5 mmol, Aldrich) dissolved in 4 mL of methanol. The solution isstirred at room temperature for 2 h after which, 77 mg of(2,3,5,6)-fluoro-4-mercaptophenylboronic acid (0.5 mmol) in 1 mL ofmethanol is added. The resulting solution is stirred for an additional 2h at room temperature. Methanol is removed under vacuo and the residueis re-dissolved in 2 mL of dichloromethane. 18 mL of diethyl ether isadded to the dichloromethane solution and the mixture is allowed to sitfor 1 h. The resulting precipitate is collected via centrifugation,washed several times with diethyl ether and dried. The dried solid isre-dissolved in water, filtered with a 0.45 μm filter and dialyzed witha 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing) in ddH₂Ofor 15 h. The dialyzed solution is lyophilized to dryness.

Example 9 Synthesis of Boronic Acid-PEG₅₀₀₀-Transferrin, (9)

A targeting agent could be placed at the other end of the PEG from theboronic acid in the compounds of Examples 5-8, for example according toan approach schematically illustrated in FIG. 12 with reference toattachment of transferrin.

Thus, the components of a system containing nucleic acids as thetherapeutic could be (targeting ligand could be a protein liketransferrin (FIG. 12), an antibody or antibody fragment, a peptide likeRGD or LHRH, a small molecule like folate or galactose, etc.). A boronicacid PEGylated targeting agent can be synthesized as follows.

In particular, to synthesize the Boronic Acid PEG₅₀₀₀-Transferrinaccording to the approach schematically illustrated in FIG. 12 thefollowing procedure was performed. A solution of 10 mg (0.13 μmol) ofHuman holo-Transferrin (iron rich) (Sigma Aldrich) in 1 mL of 0.1M PBSbuffer (p.H. 7.2) was added to 3.2 mg of OPSS-PEG₅₀₀₀-SVA (5 eq, 0.64μmol, LaysanBio Inc.). The resulting solution was stirred at roomtemperature for 2 h. The PEGylated Transferrin was purified from theunreacted OPSS-PEG₅₀₀₀-SVA using an Ultracel 50,000 MWCO (AmiconUltra-4, Millipore) and from unreacted Transferrin using a gelfiltration column G3000SWx1 (Tosoh Biosep) (confirmed by HPLC andMALDI-TOF analysis). 100 μg of the OPSS-PEG₅₀₀₀ PEGylated Transferrin in100 μL was then incubated at room temperature with 20 μL,4-mercaptophenylboronic acid (1 μg/μL, 20 μg, 100 eq.) for 1 h. Afterincubation, the solution was dialyzed twice with a YM-30,000 NMWI device(Millipore) to remove excess 4-mercaptophenylboronic and thepyridyl-2-thione by-product.

Example 10 Formulation of MAP-Nucleic Acid Particles—Gel RetardationAssay

As diagramed in FIG. 1, 1 μg of plasmid DNA or siRNA in DNAse and RNASefree water (0.1 μg/μL, 10 μL) was mixed with 10 μL of MAP at variousconcentrations in DNAse and RNASe free water to give charge ratios (“+”charge on polymer to “−” charge on nucleic acid) of 0.5, 1, 1.5, 2, 2.5,and 5. The resulting mixtures were incubated for 30 minutes at roomtemperature. 10 μL of the 20 μL solutions were loaded onto a 1% agarosegel with 3.5 μL of loading buffer and the gel was electrophoresed at 80V for 45 minutes as shown in FIGS. 3 and 4. Nucleic acid that is notcontained within the nanoparticle will migrate on the gel. These resultsgive guidance to the charge ratios necessary for nucleic acidcontainment within the nanoparticles.

Example 11 Particle Size and Zeta potential of MAP-Nucleic AcidParticles

1 μg of plasmid DNA in DNAse and RNASe free water (0.1 μg/μL, 10 μL) wasmixed with 10 μL of MAP at various concentrations in DNAse and RNASefree water to give charge ratios of 0.5, 1, 1.5, 2, 2.5, and 5. Theresulting mixtures were incubated for 30 minutes at room temperature.The 20 μL mixture was then diluted with DNAse and RNASe free water to 70μL for particle size measurements. This 70 μL solution was then dilutedto 1400 μL with 1 mM KCl for zeta potential measurements. The particlesize and zeta potential measurements were made on a ZetaPals dynamiclight scattering (DLS) instrument (Brookhaven Instruments). The resultsare shown in FIG. 5.

Example 12 Particle Size Stabilization by PEGylation with Boronic AcidPEG_(5k)

As diagrammed in FIG. 2, 2 μg of plasmid DNA in DNAse and RNASe freewater (0.45 μg/μL, 4.4 μL) was diluted to 80 μL in DNAse and RNASe freewater. This plasmid solution was mixed with 4.89 μg of MAP (0.5 μg/μL,9.8 μL) also diluted to 80 μL in DNAse and RNASe free water to give a3+/− charge ratio and a final plasmid concentration of 0.0125 μg/μL. Theresulting mixture was incubated for 30 minutes at room temperature. Tothis solution was added 480 μg of boronic acid PEG_(5K), (compound 6;Example 6), (20 μg/μL, 24 μL). This mixture was then incubated furtherfor 30 minutes, dialyzed twice in DNAse and RNASe free water with a 0.5mL 100,000 MWCO membrane (BIOMAX, Millipore Corporation) andreconstituted in 160 μL of DNAse and RNASe free water. Half of thesolution was diluted with 1.4 mL of 1 mM KCl for zeta potentialmeasurements (FIG. 6). Note that the zeta potential of the BA containingnanoparticles show a lower zeta potential than the nanoparticles that donot. These results support the conclusion that the BA containingnanoparticles have the BA localized on the exterior of thenanoparticles. The other half was used to measure the particle size. Theparticle size was measured every minute for 5 minutes after which, 10.2μL of 10×PBS was added such that the final 90.2 μL solution was in1×PBS. The particle size was then measured again every minute foranother 10 minutes as shown in FIG. 7. The BA containing nanoparticlesseparated from non-particle components (by filtration) are stable in PBSwhile the particles without the BA are not. These data support theconclusion that the BA containing nanoparticles have the BA localized ontheir exterior as they are stabilized against aggregation in PBS.

Example 13 Transfection of MAP/pDNA Particles into HeLa Cells

HeLa cells were seeded at 20,000 cells/well in 24 well plates 48 h priorto transfection and grown in medium supplemented with 10% FBS. MAPparticles were formulated to contain 1 μg of pGL3 in 200 μL of Opti-MEMI at various charge ratios of polymer to pDNA (refer to Example 9).Growth medium was removed, cells washed with PBS and the particleformulation was added. The cells were subsequently incubated at 37° C.and 5% CO₂ for 5 h before the addition of 800 μL of growth mediumsupplemented with 10% FBS. After 48 h of incubation, a fraction of thecells were analyzed for cell viability using an MTS assay. The remainingcells were lysed in 100 μL of 1× Luciferase Cell Culture Lysis Reagent.Luciferase activity was determined by adding 100 μL of Luciferase AssayReagent to 10 ul of cell lysate and bioluminescence was quantified usinga Monolight luminometer. Luciferase activity is subsequently reported asrelative light units (RLU) per 10,000 cells. Results are shown in FIG. 8and FIG. 9.

Example 14 Co-Transfection of MAP/pDNA and/or siRNA Particles into HeLaCells

HeLa cells were seeded at 20,000 cells/well in 24 well plates 48 h priorto transfection and grown in medium supplemented with 10% FBS. MAPparticles were formulated to contain 1 μg of pGL3 and 50 nM of siGL3 in200 μL of Opti-MEM I at a charge ratio of 5+/−. Particles containingonly pGL3 or pGL3 and siCON were used as controls. Growth medium wasremoved, cells washed with PBS and the particle formulation was added.The cells were subsequently incubated at 37° C. and 5% CO₂ for 5 hbefore the addition of 800 μL of growth medium supplemented with 10%FBS. After 48 h of incubation, the cells were assayed for Luciferaseactivity and cell viability as described in Example 12. Results areshown in FIG. 10. Since the RLU is lowered in transfections with thesiGL3 (correct sequence), both the siGL3 and the pGL3 must beco-delivered.

Example 15 Transfection of MAP/siGL3 into HeLa-LUC Cells

HeLa-LUC cells (contain gene encoding for the firefly luciferaseprotein) were seeded at 20,000 cells/well in 24 well plates 48 h priorto transfection and grown in medium supplemented with 10% FBS. MAPparticles were formulated to contain 50 and 100 nM siGL3 in 200 μL ofOpti-MEM I at a charge ratio of 5+/−. Growth medium was removed, cellswere washed with PBS and the particle formulation was added. The cellswere subsequently incubated at 37° C. and 5% CO₂ for 5 h before theaddition of 800 μL of growth medium supplemented with 10% FBS. After 48h of incubation, the cells were assayed for Luciferace activity and cellviability as described in Example 12. Results are shown in FIG. 11.Since the RLUs decline with increasing concentration of siGL3, thesedata suggest that inhibition of an endogenous gene can occur.

Example 16 Synthesis of Mucic Acid Diiodide, (10)

1 g (2.7 mmol) of mucic acid diamine (Example 3) was mixed with 3.8 mL(27.4 mmol) of triethylamine and 50 mL of anhydrous DMF prior to thedropwise addition of 1.2 mL (13.7 mmol) iodoacetylchloride in a 250 mLround bottom flask. This mixture was allowed to react overnight underconstant stirring at room temperature. The solvent was subsequentlyremoved by vacuum pump, the product filtered, washed with methanol anddried under vacuum to yield 0.8 g (1.3 mmol, 46%) of mucic aciddiiodide. ¹H NMR ((CD₃)₂SO) δ 8.20 (s 2H), 2H), 7.77 (s, 2H), 4.11 (m,2H), 4.03 (m, 2H), 3.79 (m, 2H), 3.11-3.17 (m, 2H), 1.78 (d, 2H). ESI/MS(m/z): 652.8 [M+Na]⁺

Example 17 Synthesis of Mucic Acid Dicysteine, (11)

To 7 mL of 0.1 M degassed sodium carbonate was added 17 mg of L-cysteineand 0.4 g of mucic acid diiodide. The resulting suspension was broughtto reflux at 150° C. for 5 h until the solution turned clear. Thismixture was then cooled to room temperature and adjusted to pH 3 via 1 NHCl. Slow addition of acetone was then employed for productprecipitation. After filtration, washing with acetone and vacuum drying,60 mg of crude product was obtained.

Example 17 Synthesis of Mucic Acid Dicysteine, (11)

To 20 mL of 0.1 M degassed sodium phosphate buffer at pH 7.5 in a 50 mLround bottom flask was added 0.38 g of L-cysteine (3.2 mmol) and 0.40 g(0.6 mmol) of mucic acid diiodine. The resulting suspension was allowedto reflux at 75° C. overnight, cooled to room temperature andlyophilized. 80 mL of DMF was subsequently added to this lyophilizedlight brown powder and separation of the insoluble excess reagent andphosphate salts from the soluble product was achieved by filtration. DMFwas removed under reduced pressure and the product was vacuum dried togive 12 mg (0.02 mmol, 3%) of mucic acid dicysteine.

Example 18 Polymer Synthesis, (Poly(Mucic Acid-DiCys-PEG)) (12)

12 mg (21.7 μmol) of mucic acid dicysteine and 74 mg (21.7 μmol) ofPEG-DiSPA 3400 were dried under vacuum prior to the addition of 0.6 mLof anhydrous DMSO under argon in a 2 neck 10 mL round bottom flask.After 10 min of stirring, 9 μL (65.1 μmol) of anhydrous DIEA wastransferred to the reaction vessel under argon. This mixture was stirredunder argon overnight. The polymer containing solution was then dialyzedusing a 10 kDa membrane centrifugal device and lyophilized to yield 47mg (58%) of Poly(Mucic Acid-DiCys-PEG).

This polymer containing polyols is an anionic AB polymer.

Example 19 Covalent Attachment of Drug (Camptothecin, CPT) to Mucic AcidPolymer, (13)

10 mg (2.7 μmol of repeat units) of Poly(Mucic Acid-DiCys-PEG) wasdissolved in 1.5 mL of anhydrous DMSO in a glass jar. After stirring for10 min, 1.1 μL of DIEA (6.3 μmol), 3.3 mg (6.3 μmol) of TFA-Gly-CPT, 1.6mg (8.1 μmol) of EDC and 0.7 mg (5.9 μmol) of NHS were added to thereaction mixture. After stirring for 8 hrs, 1.5 mL of ethanol was addedand the solvents were removed under reduced pressure. The precipitatewas dissolved in water and insoluble materials were removed byfiltration through a 0.2 μm filter. The polymer solution was thendialyzed against water via a 10 kDa membrane and subsequentlylyophilized to give the Poly(Mucic Acid-DiCys-PEG)-CPT conjugate.

Example 20 Formulation of Nanoparticle with CPT-Mucic Acid Polymer (13)in Water, (20)

The Effective diameters of poly(Mucic Acid-DiCys-PEG) and poly(MucicAcid-DiCys-PEG)-CPT conjugate were measured by formulating the polymersin double distilled water (0.1-10 mg/mL) and evaluated via dynamic lightscattering (DLS) using a ZetaPALS (Brookhaven Instrument Co) Instrument.3 successive runs of 1 min each were subsequently recorded and averaged.The zeta potentials of both compounds was measured in a 1.1 mM KClsolution using a ZetaPALS (Brookhaven Instrument Co) Instrument. 10successive automated runs at target residuals of 0.012 were thenperformed and results averaged (FIG. 14). In particular, there twodistributions were measured for the poly(Mucic Acid-DiCys-PEG)-CPTconjugate the predominant distribution was a 57 nm (60% of the totalparticle population). A second minor distribution was also was measuredat 233 nm.

Example 21 Formulation of Boronic Acid-PEGylated Nanoparticle withCPT-Mucic Acid Polymer (13) and Boronic Acid-Disulfide-PEG₅₀₀₀ (6) inWater

The boronic acid PEGylated poly(Mucic Acid-DiCys-PEG)-CPT nanoparticleis formulated by dissolving the polymer in double distilled water at aconcentration of 0.1 mg/mL followed by the addition of Polymer 6(BA-PEG) also in water, such that the ratio of PBA-PEG to the diols onthe mucic acid sugar in the poly(Mucic Acid-DiCys-PEG)-CPT conjugate is1:1. The mixture is incubated for 30 mins after which the effectivediameter and zeta potential are measured using a ZetaPALS (BrookhavenInstrument Co) instrument.

Example 22 Targeted Nanoparticles for pDNA Delivery in Mice

The plasmid pApoE-HCRLuc contains the gene to express luciferase and isunder the control of a liver specific promoter. Polymer (MAP) 4 (0.73mg), polymer 6 (73 mg) and polymer 9 (0.073 mg) were combined in 5 mL ofwater and then 1.2 mL of water containing the pApoE-HCRLuc plasmid wereadded (gives a charge ratio of polymer 4 to the plasmid of +3). Theparticles were placed in D5W (5% glucose in water) by successive spinfiltering with subsequent additions of D5W (starting from the initialformulation that was in water). Nude mice were implanted with Hepa-1-6liver cancer cells and tumors were allowed to grow until a size ofapproximately 200 mm³. Injections of the targeted nanoparticles weredone i.v. in the tail vein at an amount equal to 5 mg plasmid/kg mouse.The mice were imaged 24 hours after the injections. The mice showed nosigns of toxicity and there was luciferase expression detected in theregion of the tumor and not in the region of the liver.

Example 23 Synthesis of PBA-PEG and nitroPBA-PEG and Determination ofpKa

To prepare a stabilized and targeted nanoparticle, the covalent,reversible binding property between boronic acids and MAP (diolcontaining) was used. The pKa of phenylboronic acid (PBA) is high at8.8. To decrease the pKa, PBAs with electron withdrawing groups on thephenyl ring were employed to increase the acidity of the boron atom.Commercially available 3-carboxyPBA and 3-carboxy 5-nitroPBA wereconverted into acyl chlorides using oxalyl chloride. These acyl chloridespecies were then reacted with NH₂—PEG to form PBA-PEG and nitroPBA-PEGrespectively. The addition of bases DMF and DIPEA were required for thesynthesis reactions to proceed. However, the presence of these basesresulted in tetrahedral adduct formations with acidic PBA. To removethese adducts, work up in 0.5 N HCl with subsequent equilibration toneutral pH by dialysis against water was carried out.

For synthesis of PBA-PEG 200 mg (1.21 mM) of 3-carboxyphenylboronic aciddissolved in 5 ml of anhydrous tetrahydrofuran was added 18.7 μl (0.24mM) of anhydrous dimethylformamide under argon. This reaction vessel wastransferred into an ice bath and 195 μl (2.89 mM) of oxalyl chloride wasslowly added under argon. The reaction was allowed to proceed under ventand constant stirring for 2 h at room temperature. Solvent and excessreagent were removed under a vacuum. 37 mg (0.2 mM) the resulting driedacyl chloride compound was dissolved in 15 ml of anhydrousdichloromethane under argon. Then 500 mg (0.1 mM) of NH2-PEG5 kDa-CO2Hand 52 μl (0.3 mM) of dry DIPEA under argon were added. After overnightreaction under constant stirring, solvent was removed under a vacuum andthe dried product was reconstituted in 0.5 N HCl. This solution waspassed through a 0.2 μm filter (Acrodisc®) and dialyzed against waterwith a 3 kDa MWCO membrane filter device (Amicon™) until constant pH wasattained. The supernatant was then filtered with a 0.2 μm filter(Acrodisc®) and lyophilized to yield 377 mg (73%) of PBA-PEG-CO2H. 1HNMR ((CD3)2SO) δ 12.52 (s, 1H), 8.39 (t, 1H), 8.22 (s, 1H), 8.14 (s,2H), 7.88 (d, 1H), 7.82 (d, 1H), 7.39 (t, 1H), 3.99 (s, 2H), 3.35-3.62(PEG peak). MALDI-TOF (m/z): 5600 g/mol (NH2-PEG5 kDa-CO2H, 5400 g/mol).

Synthesis of nitroPBA-PEG-CO2H was performed in the same way as forPBA-PEG-CO2H except the starting material was 3-carboxy5-nitrophenylboronic acid. The reaction yielded 393 mg (76%) ofnitroPBA-PEG-CO2H. 1H NMR ((CD3)2SO) δ 12.52 (s, 1H), 8.89 (t, 1H), 8.72(s, 1H), 8.68 (s, 1H), 8.64 (s, 1H), 8.61 (s, 2H), 3.99 (s, 2H),3.35-3.62 (PEG peak). MALDI-TOF (m/z): 5600 g/mol (NH2-PEG5 kDa-CO2H,5400 g/mol).

The pKa of the synthesized PBA compounds were found by measuring thechange in absorbance of PBA as it converted from trigonal (in low pH) totetrahedral (in high pH) conformation. To a solution of 10-3 M PBA in0.1 M PBS, pH 7.4 was titrated 1 N NaOH. pH was recorded andcorresponding samples were removed for absorbance measurements at 268nm.

TABLE 2 pKa and binding constants of PBA compounds Binding constantCompound pKa with MAP (M⁻¹) PBA 8.8 20 PBA—PEG—CO₂H 8.3 520nitroPBA—PEG—CO₂H 6.8 1420

Example 24 Antibody Conjugation to nitroPBA-PEG

Following production of nitroPBA-PEG the resulting compound wasconjugated to an antibody. For conjugation 36 mg (6.4 μM) ofnitroPBA-PEG-CO2H, 12.3 mg (64 μM) of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 11.1 mg (96 μM)of NHS were dissolved in 2.4 ml of 0.1 M MES buffer, pH 6.0. Thismixture was reacted for 15 min on a rotating shaker at room temperature.Excess reactants were dialyzed away with 3 kDa molecular weight cutoffmembrane filter device (Amicon™). This activated carboxylic acid PEGcompound was added to 20 mg (0.14 mM) of human IgG1 antibody(Herceptin®) in 0.1 M PBS, pH 7.2. The reaction was carried out on arotating shaker at room temperature for 2 h and then dialyzed 4 timesusing a 50 kDa molecular weight cutoff membrane filter device (Amicon™)against 1×PBS at pH 7.4. Analysis of the conjugate by mass spectrometry(MALDI-TOF) showed an average conjugation of 1 to 2 nitroPBA-PEGcompounds per antibody.

Example 26 Formation and Characterization of Targeted MAP Nanoparticles

The effect of PBA-PEG and nitroPBA-PEG on nanoparticle formation andstability were examined. A solution of PBA-PEG was added to a solutionof MAP at 3+/− charge ratio (positive charge from MAP for every negativecharge from siRNA) in phosphate buffer at pH 7.4. This mixture wasallowed to sit at room temperature for 10 min for the complexation ofPBA-PEG with the diol-containing MAP. This was then added to an equalvolume of siRNA in water and mixed by pipetting. After 10 min, tests andcharacterizations were performed. The formulations of particlesstabilized with nitroPBA-PEG and at different charge ratios wereconducted in the same manner. The conjugation of PBA-PEG formedMAP/siRNA particles of 150-300 nm in size. MAP-4 (indicating 4 methyleneunits between charge center (amidine) and binding site (polyol))resulted in smaller nanoparticles than when MAP-2 was used. However,upon the addition of salt, all particles aggregated indicating thatPBA-PEG was incapable of providing sufficient stability to thenanoparticles. When nitroPBA-PEG was used, particles at all chargeratios tested, 1 and 3 (+/−), demonstrated stabilized nanoparticles. Thedifference in particle stability conferred by PBA-PEG and nitroPBA-PEGindicates the importance of modifying the PEGylated boronic acidcompounds to possess stronger binding to polyols.

For siRNA condensed with MAP-4 without the presence of stabilizing agentPEG, a particle size of 640 nm was observed with a high zeta potentialof +32 mV. When nitroPBA-PEG was added to stabilize the particle, thediameter reduced to 130 nm with a negative surface charge of −4 mV. Thisnegative charge is a result of the binding between boronic acid anddiols. When 0.25 mol % IgG (Herceptin®) conjugated nitroPBA-PEG wasintroduced, interestingly the particle size reduced further to 82 nmwhile the zeta potential remained similar.

The salt stability for MAP-4/siRNA nanoparticles with various PEG groupsattached was assessed. Particle size was recorded for 5 runs at 1 minusing a zeta potential analyzer (DLS ZetaPALS instrument, BrookhavenInstruments, Holtsville, N.Y.). Measurement was stopped to allow theaddition of a 10×PBS solution to the particle formulation such that theresulting particle formulations contain 1×PBS. Measurements weresubsequently restarted and 10 successive runs of 1 min each wererecorded to study the effect of salt addition on particle stability. Asshown in FIG. 16, without the presence of a stabilizing agent PEG, theparticles aggregated. When nitroPBA-PEG was present, particles remainedstable in salt conditions.

Example 27 Formation and Characterization of Targeted MAP-CPTNanoparticles

To prepare a targeted MAP-CPT nanoparticle, the covalent, reversiblebinding property between boronic acids and MAP (diol containing) wasused. The binding constant between PBA and MAP is low at about 20 M⁻¹.To increase the binding constant, PBAs with electron withdrawing groupson the phenyl ring were employed to increase the acidity of the boronatom and thus elevate the binding constant with MAP. Commerciallyavailable 3-carboxyPBA and 3-carboxy 5-nitroPBA were converted into acylchlorides using oxalyl chloride. These acyl chloride species were thenreacted with NH2-PEG-CO₂H to form PBA-PEG-CO₂H and nitroPBA-PEG-CO₂H,respectively. The addition of bases DMF and DIPEA were required for thesynthesis reactions to proceed. However, the presence of these basesresulted in tetrahedral adduct formations with acidic PBA. To removethese adducts, work up in 0.5 N HCl with subsequent equilibration toneutral pH by dialysis against water was carried out.

pKa values of the modified PBAs were determined by absorbance changesdue to conformational change of PBA from trigonal to tetrahedral form asthe pH was increased (Table 2). PBA with electron withdrawing groupsresulted in lower pKa's, with nitroPBA-PEG-CO₂H having the lowest pKa of6.8. Thus, at physiological pH, most of the PBA was present in thereactive anionic tetrahedral form. Binding constants between PBA and MAPwere found by competitive binding with Alizarin Red S in 0.1 M PBS, pH7.4. Because of the low pKa value and high binding constant with MAPobserved for nitroPBA-PEG-CO₂H, it was chosen as the linker between IgG(Herceptin®) and MAP-CPT nanoparticles.

The conjugation reaction between IgG (Herceptin®) and nitroPBA-PEG-CO₂Hproceeded via EDC coupling (described above). An average of 1 to 2 PEGswere attached per IgG (Herceptin®) antibody.

TABLE 3 Characterization of MAP, MAP—CPT conjugate, MAP—CPTnanoparticles and targeted MAP—CPT nanoparticles Short Medium Long MAPBase added (equivalent)^(a) 1.1 1.6 2 MW^(b) (kDa) 20 65 102Polydispersity^(c) 1.22 1.36 1.13 # repeat units (n) 5-7 15-21 25-30MAP—CPT MW^(b) (kDa) 22 75 114 conjugate wt % CPT conjugated 9.8 12.710.1 MAP—CPT # conjugates/particle 2-3 2-3 2-3 nanoparticle #CPT/particle ~14 ~60 ~72 particle size (nm) ~30 ~30 ~30 zeta potential(mV) −1.3+/−0.6 −0.5+/−0.5 −0.8+/−0.5 Targeted # Herceptin/particle 1MAP—CPT particle size (nm) ~40 nanoparticle zeta potential (mV)−0.4+/−0.6 ^(a)Equivalent amount of N, N-diisopropylethylamine (DIPEA)added per amine group in mucic acid di(Asp—amine). ^(b)MW, molecularweight determined as (Mw + Mn)/2; Mw, weight average molecular weight;Mn, number average molecular weight. ^(c)Polydispersity determined asMw/Mn.

An average size of about 40 nm was observed for targeted MAP-CPTnanoparticles by DLS and cryo-EM (Table 3). This increase in about 10 nmfrom the non-targeted nanoparticle suggests the attachment of IgG(Herceptin®) to the nanoparticle (the amount was about one Herceptin®antibody per nanoparticle). Indeed, from cryo-EM images, the targetednanoparticles were not spherical but appeared to have protrusionsindicating the attachment of antibodies. The surface charge of targetedMAP-CPT nanoparticles was slightly higher than MAP-CPT nanoparticles dueto the presence of a positively charged Herceptin at pH 7.4 (pI ofHerceptin®, 9.2).

Example 28 Cellular Uptake Studies

A HER2 overexpressing human breast cancer cell line (BT-474), was usedto examine the cellular uptake of Herceptin® targeted MAP-CPTnanoparticles versus the non-targeted nanoparticles. A HER2 negativebreast cancer cell line (MCF-7), was used as a negative control. Forthese studies 24 well plates were seeded with either BT-474 (inRPMI-1640 medium supplemented with 10% fetal calf serum) or MCF-7 (inDulbecco's Modified Eagle medium supplemented with 10% fetal calf serum)(Cellgro, Manassas, Va.) at 20,000 cells per well and kept at 37° C. ina humidified oven with 5% CO₂. After 40 h, media were replaced with 0.3ml of fresh media containing either medium MAP-CPT (40 μg CPT/ml),medium MAP-CPT with free Herceptin® at 10 mg/ml, targeted MAP-CPT (40 μgCPT/ml) at varying targeting densities or targeted MAP-CPT with freeHerceptin® at 10 mg/ml. Transfection was conducted for 30 min at 37° C.Formulations were then removed and cells washed twice with cold PBS. 200μl of RIPA buffer was added per well for cell detachment and lysis.Lysed cells were then incubated at 4° C. for 15 min and centrifuged at14,000 g for 10 min at 4° C. A portion of the supernatant was used forprotein quantification via BCA assay. To another portion was added anequal amount of 0.1 N NaOH, this was incubated at room temperatureovernight and fluorescence was measured at 370/440 nm using knownconcentrations of MAP-CPT as standard.

It was observed that one Herceptin®-PEG-nitroPBA per nanoparticle wassufficient to achieve about 70% greater uptake in BT-474 cell linecompared to the non-targeted nanoparticle (FIG. 17A). Uptake of thetargeted MAP-CPT in BT-474 cells showed inhibition in the presence offree Herceptin® (FIG. 17B). In contrast, uptake of MAP-CPT nanoparticlesin MCF-7 exhibited no dependence on targeting or on presence of freeHerceptin® (FIG. 2B). These data indicate that there is receptormediated uptake in the BT-474 cells by the targeted MAP-CPTnanoparticles via engagement of the HER2 receptor. Cellular uptake ofboth the targeted and non-targeted MAP-CPT nanoparticles also occurredvia nonspecific fluid phase endocytosis.

Example 29 In Vitro Release Studies

Experiments were conducted to assess the release of CPT from short,medium, long MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles.These studies were conducted using 0.32 mg CPT/ml in 1×PBS at pH 6.5, 7or 7.4; BALB/c mice plasma, human plasma, and 1×PBS at pH 7.4 containing3 mg/ml of low density lipoprotein (LDL), or 100 units/ml ofButyrylcholinesterase (BCHE) or a combination of LDL and BCHE.

Media pipetted into 96 well plates were incubated at 37° C. in ahumidified oven for 2 h for equilibration. Formulations were mixed intothe relevant media and placed back into the oven. Samples were taken outat predetermined time points and immediately frozen at −80° C. untiltime for analysis. For release in BALB/c mice plasma and in humanplasma, incubation was carried out in a humidified oven at 37° C. with5% CO₂ to maintain the carbonic acid/bicarbonate buffer system, themajor pH buffer system in plasma at physiological pH levels.

The amount of unconjugated CPT was determined by first mixing 10 μl ofsample with 10 μl of 0.1 N HCl and incubating at room temperature for 30min. 80 μl of methanol was then added and the mixture incubated at roomtemperature for 3 h for protein precipitation. This mixture wascentrifuged at 14,000 g for 10 min at 4° C., supernatant was filteredwith a 0.45 μm filter (Millex-LH), diluted 20 folds with methanol and 10μl of the resulting solution injected into HPLC. The peak area of theeluted CPT (at 7.8 min) was compared to that of control. To measure thetotal amount of CPT, 10 μl of sample was mixed with 6.5 μl of 0.1 NNaOH. This solution was incubated at room temperature for 1 h for CPT tobe released from parent polymer. 10 μl of 0.1 N HCl was then added toconvert the carboxylate CPT form to the lactone form. 73.5 μl ofmethanol was subsequently added and mixture incubated for 3 h at roomtemperature. The sample was then centrifuged and processed as above.Polymer-bound CPT concentration was determined from the differencebetween total CPT and unconjugated CPT concentrations.

Release of CPT from the short, medium and long MAP-CPT nanoparticles andfrom the targeted MAP-CPT nanoparticles all exhibited first-orderkinetics. Release half-lives of CPT from the short, medium and longMAP-CPT nanoparticles were very similar in all conditions tested, whilelonger half-lives for the targeted MAP-CPT were observed (Table 4). Astrong dependence of release rate with pH was observed. As the pHincreased from 6.5 to 7.4, the release half-lives reduced from 338 h to58 h for the short, medium and long MAP-CPT nanoparticles. These dataindicate that hydrolysis plays an important role in the release of CPT.A release half-life of 59 h was observed in BALB/c mice plasma, while asignificantly lower half-life of 38 h was obtained in human plasma forthe short, medium and long MAP-CPT nanoparticles. It is known that miceplasma contains more esterase activity than human plasma, while humanplasma contains more “fat” than mouse plasma. Therefore, to understandthe differences in release rates between human and mice plasma, thecontributions of esterase and fat to CPT release rates were individuallytested. Butyrylcholinesterase (BCHE) is present in both mice and humanplasma and was used to test esterase contribution to the release rate.Low density lipoprotein (LDL) was chosen to test the contribution of“fat” to the release rate. These components were constituted in PBS (pH7.4). It was found that the presence of BCHE did not affect release rate(half-life of 61 h compared to 58 h in PBS, pH 7.4, for the short,medium and long MAP-CPT nanoparticles). However, the addition of LDLdramatically increased the release rates. This effect is likely due tonanoparticle disruption by competing hydrophobic interactions.Therefore, nanoparticle disruption by the presence of “fat”, and thesubsequent CPT cleavage by hydrolysis appear to be another mainmechanism of CPT release from MAP-CPT nanoparticles. In vitro releasefrom the Herceptin® targeted MAP-CPT nanoparticles shows longerhalf-lives than the non-targeted versions. It is possible that thepresence of Herceptin® with a pI of 9.2 increased the stability of thenegatively charged nanoparticles by electrostatic interactions, and thusshielded the nanoparticles from some of the competing hydrophobicinteractions.

TABLE 4 In vitro release half-lives (t_(1/2)) of CPT from short, mediumand long MAP—CPT nanoparticles and from targeted MAP—CPT nanoparticlesin various media PBS t_(1/2)(h) pH 6.5 pH 7 pH 7.4 Short, medium, long338 178 58 MAP—CPT Targeted MAP—CPT 396 204 78 Plasma t_(1/2)(h) MiceHuman Short, medium, long 69 38 MAP—CPT Targeted MAP—CPT 63 46 PBS pH7.4 t_(1/2)(h) LDL^(a) BCHE^(b) LDL + BCHE^(c) Short, medium, long 45 6144 MAP—CPT Targeted MAP—CPT 62 76 62 Abbreviations: PBS, phosphatebuffered saline; LDL, low density lipoprotein; BCHE,Butyrylcholinesterase. ^(a)PBS, pH 7.4, containing 3 mg/ml LDL. ^(b)PBS,pH 7.4, containing 100 units/ml BCHE. ^(c)PBS, pH 7.4, containing 3mg/ml LDL and 100 units/ml BCHE.

Example 30 Cytotoxicity Assays

In vitro cytotoxicities of medium MAP, nitroPBA-PEG, CPT, medium MAP-CPTnanoparticles, targeted MAP-CPT nanoparticles and Herceptin® wereevaluated in two HER2+ breast cancer cell lines (BT-474, SKBR-3) and twoHER2− breast cancer cell lines (MCF-7, MDA-MB-231) (Table 5). For thesestudies cells were kept at 37° C. in a humidified oven with 5% CO₂.BT-474, MCF-7, SKBR-3 and MDA-MB-231 cell lines were incubated inRPMI-1640 medium, Dulbecco's Modified Eagle medium, McCoy's 5A Modifiedmedium and Dulbecco's Modified Eagle medium respectively (allsupplemented with 10% fetal calf serum). 3,000 cells per well wereplated into 96 well plates. After 24 hours, media was removed andreplaced with fresh media containing different concentrations of mediumMAP, nitroPBA-PEG, CPT, medium MAP-CPT nanoparticles, targeted MAP-CPTnanoparticles or Herceptin®. After 72 h of incubation, formulations werereplaced with fresh media, and 20 μl of CellTiter 96® AQueous OneSolution cell proliferation assay (Promega) was added per well. This wasincubated for 2 hours in a humidified oven at 37° C. with 5% CO₂ beforeabsorbance measurements at 490 nm. Wells containing untreated cells wereused as controls.

Medium MAP and nitroPBA-PEG gave IC₅₀ values of above 500 μM and 1000 μM(highest concentrations tested), respectively, indicating minimaltoxicity. IC₅₀ concentrations of CPT, medium MAP-CPT nanoparticles andtargeted MAP-CPT nanoparticles were based on the content of the CPT. Itis noted that CPT was released gradually from medium MAP-CPTnanoparticles and targeted MAP-CPT nanoparticles (see In Vitro ReleaseStudies). Additionally, nanoparticle uptake by cells results in longrelease times due to the acidic nature of the endosomes. These factorscontribute to the observed higher IC₅₀ values for the medium MAP-CPTnanoparticles and targeted MAP-CPT nanoparticles compared to CPT (Table5). In HER2+ cell lines, the targeted MAP-CPT nanoparticles gave lowerIC₅₀ values compared to MAP-CPT alone, while in HER2− cell lines,targeting did not affect the IC₅₀. BT-474 was the most resistant cellline to CPT, and consequently to medium MAP-CPT nanoparticles. Theaddition of Herceptin at concentrations of 0.001-0.5 μM to BT-474 orSKBR-3 cells resulted in a constant cell viability of about 60% ascompared to a no treatment control. The lack of a true IC₅₀ value overthis concentration range for these cell lines has been observedpreviously (Phillips, et al., Cancer Res. 68, 9280-9290 (2008)). Therewere no effects observed in HER2− cell lines at Herceptin concentrationsof up to 0.5 μM.

TABLE 5 IC₅₀ values of medium MAP, nitroPBA—PEG, CPT, medium MAP—CPTnanoparticles, targeted MAP—CPT nanoparticles and Herceptin for a rangeof breast cancer cell lines. MCF-7 MDA-MB-231 SKBR-3 BT-474 HER2expression − − + + IC₅₀ Medium MAP >500 >500 >500 >500 (μM)nitroPBA—PEG >1000 >1000 >1000 >1000 CPT 0.3 0.1 0.03 4 Medium 0.5 0.60.2 40 MAP—CPT Targeted 0.5 0.6 0.1 6 MAP—CPT Herceptin no effect noeffect no value^(a) no value^(a) ^(a)no value, IC₅₀ value was notobtained over the concentration range of 0.001-0.5 μM.

Example 31 Pharmacokinetic Studies

Plasma pharmacokinetic studies of short, medium, long MAP-CPTnanoparticles and targeted MAP-CPT nanoparticles at 10 mg/kg (CPT basis)injections were conducted in female BALB/c mice (FIG. 18). For thesestudies nanoparticle formulated in 0.9 wt % NaCl (non-targetednanoparticles) or PBS (targeted MAP-CPT nanoparticles) were administeredvia bolus tail vein injection into 12-16 weeks old female BALB/c mice.At predetermined time points, blood was collected via saphenous veinbleed with blood collection tubes (Microvette CB 300 EDTA, Sarstedt).Samples were immediately centrifuged at 10,000 g, 4° C. for 15 minutesand supernatant removed and stored at −80° C. until time for analysis.Analyses for unconjugated and polymer-bound CPT were as follows. Theamount of unconjugated CPT was determined by first mixing 10 μl ofsample with 10 μl of 0.1 N HCl and incubating at room temperature for 30min. 80 μl of methanol was then added and the mixture incubated at roomtemperature for 3 h for protein precipitation. This mixture wascentrifuged at 14,000 g for 10 min at 4° C., supernatant was filteredwith a 0.45 μm filter (Millex-LH), diluted 20 folds with methanol and 10μl of the resulting solution injected into HPLC. The peak area of theeluted CPT (at 7.8 min) was compared to that of control. To measure thetotal amount of CPT, 10 μl of sample was mixed with 6.5 μl of 0.1 NNaOH. This solution was incubated at room temperature for 1 h for CPT tobe released from parent polymer. 10 μl of 0.1 N HCl was then added toconvert the carboxylate CPT form to the lactone form. 73.5 μl ofmethanol was subsequently added and mixture incubated for 3 h at roomtemperature. The sample was then centrifuged and processed as above.Polymer-bound CPT concentration was determined from the differencebetween total CPT and unconjugated CPT concentrations. Non-compartmentalmodeling software PK Solutions 2.0 by Summit Research Services(Montrose, Colo.) was used for pharmacokinetic data analysis. Allanimals were treated as per National Institute of Health Guidelines forAnimal Care and approved by California Institute of TechnologyInstitutional Animal Care and Use Committee.

Polymer bound CPT for all nanoparticles displayed a biphasic profilewith a fast redistribution phase (α) and a long elimination phase (β)(FIG. 18A). The elimination phase for medium and long MAP-CPTnanoparticles were particularly prolonged with half-lives of 16.6 and17.6 h, respectively, and gave high area under the curve (AUC) values of2298 and 3636 μg*h/ml, respectively. Additionally, they showed lowvolumes of distribution and clearance rates. 24 h after injection, 11.3%of the injected dose of the medium MAP-CPT nanoparticles and 20.5% ofthe injected dose of the long MAP-CPT nanoparticles were stillcirculating in plasma as polymer bound CPT. In contrast, mice injectedwith CPT alone at 10 mg/kg showed fast clearance, with an AUC of only1.6 μg*h/ml and 0.01% of the injected dose remaining in circulationafter 8 h. Targeting of medium MAP-CPT nanoparticles effected thepharmacokinetic profile by increasing the redistribution phase andprolonging the elimination phase to 21.2 h with a high AUC value of 2766μg*h/ml. The amounts of unbound CPT in plasma for all nanoparticles werelow at all time points (FIG. 18B).

Example 32 Determination of Maximum Tolerable Dose (MTD) in Nude Mice

MTD values were determined in female NCr nude mice and defined as thehighest dose resulting in less than 15% body weight loss and with notreatment related deaths. In these experiments 12 weeks old female NCrnude mice were randomly divided into thirteen groups containing fivemice each. Formulations medium MAP or nitroPBA-PEG at 200 mg/kg, shortMAP-CPT nanoparticles at 10, 15 or 20 mg/kg (CPT basis), medium MAP-CPTnanoparticles at 8, 10 or 15 mg/kg (CPT basis), long MAP-CPTnanoparticles at 5, 8 or 10 mg/kg (CPT basis) and targeted MAP-CPTnanoparticles at 8 or 10 mg/kg (CPT basis) were administered on day 0and day 7 via intravenous tail vein injection. All injections wereformulated in 0.9 w/v % saline except for targeted MAP-CPT which wasformulated in PBS, pH 7.4. Weight and health of the mice were recordedand monitored daily for 2 weeks after the start of the treatment. MTDwas defined as the highest dose resulting in less than 15% body weightloss and with no treatment related deaths. Animals were euthanized whencriteria for MTD was exceeded or at the end of the study by CO₂asphyxiation.

Mice treated with medium MAP, nitroPBA-PEG, short, medium and longMAP-CPT and targeted MAP-CPT were weighed and monitored for health aftertwo weekly doses on day 0 and day 7 (Table 6). The weight and health ofthe mice were unaffected by treatment at high doses of medium MAP ornitroPBA-PEG, indicating minimal toxicity for these polymericcomponents. For groups containing CPT, maximum weight loss appeared 3 to5 days after each treatment. Most of the groups gained back the lostweight. If body weight loss continued and exceeded an average of 15%,then the study was concluded. Some mice in groups treated with mediumMAP-CPT at 15 mg/kg and long MAP-CPT at 10 mg/kg showed diarrhea andappeared weak. All other mice appeared healthy. MTD values were found tobe 20, 10, 8 and 8 mg/kg (on CPT basis) for short, medium, long MAP-CPTand targeted MAP-CPT respectively (Table 6).

TABLE 6 Treatment response for maximum tolerable dose (MTD) study DoseMax % weight (mg/kg)^(a) loss (day)^(b) Death medium MAP 200 −1.1 (11) 0nitroPBA—PEG 200 −0.5 (2) 0 short MAP—CPT 10 −4.5 (2) 0 short MAP—CPT 15−6.1 (10) 0 short MAP—CPT 20 −10.2 (12) 0 medium MAP—CPT 8 −6.8 (10) 0medium MAP—CPT 10 −14.9 (12) 0 medium MAP—CPT 15 −18.4 (4) culled^(c)long MAP—CPT 5 −4.8 (4) 0 long MAP—CPT 8 −9.2 (12) 0 long MAP—CPT 10−16.5 (10) culled^(c) targeted MAP—CPT 8 −5.2 (4) 0 targeted MAP—CPT 10−15.2 (9) culled^(c) ^(a)All groups containing MAP—CPT are based on mgCPT/kg. ^(b)Maximum percent body weight loss. ^(c)Animals culled due toexceeding 15% body weight loss.

Example 33 Biodistribution in Nude Mice

To assess the biodistribution targeted nanoparticles in mice 7 weeks oldNCr nude mice were transplanted subcutaneously with 17β-estradiolpellets. After 2 days, BT-474 carcinoma cells suspended in RPMI-1640medium were injected subcutaneously into the right front flank at 10million cells/animal. Treatment began a day after the tumors reached anaverage size of 260 mm³. Animals were randomized into two groups of sixmice per group and treated via intravenous tail vein injections witheither MAP-CPT nanoparticles at 5 mg CPT/kg (in PBS, pH 7.4) or targetedMAP-CPT nanoparticles at 5 mg CPT/kg and 29 mg Herceptin®/kg (in PBS, pH7.4). After 4 h and 24 h, blood was collected from three animals fromeach group via saphenous vein bleed. Animals were then euthanized by CO₂asphyxiation and perfused with PBS. Tumor, lung, heart, spleen, kidneyand liver were harvested and sectioned into two equal sized pieces. Onepiece was embedded in Tissue-Tek® OCT (Sakura) and the other collectedin Eppendorf tubes, both were frozen immediately at −80° C. until timefor processing.

Organs were weighed and 100 mg of each were placed in Lysing Matrix Ahomogenizer tubes containing an added ¼inch ceramic sphere (MPBiomedicals, Solon, Ohio). 1 ml of RIPA lysis buffer (Thermo Scientific)was added and tissues were homogenized using a FastPrep®-24 homogenizer(MP Biomedicals, Solon, Ohio) at 6 m/s for 30 s. This was repeated for 3times with 1 min of cooling on ice in between each round. Samples werethen centrifuged at 14,000 g for 15 min at 4° C. The amount ofunconjugated CPT was determined by first mixing 10 μl of the supernatantwith 10 μl of 0.1 N HCl and incubating at room temperature for 30 min.80 μl of methanol was then added and the mixture incubated at roomtemperature for 3 h for protein precipitation. This mixture wascentrifuged at 14,000 g for 10 min at 4° C., supernatant was filteredwith a 0.45 μm filter (Millex-LH) and 10 μl of the resulting mixtureinjected into HPLC. The peak area of the eluted CPT (at 7.8 min) wascompared to that of control. To measure the total amount of CPT, 10 μlof sample was mixed with 6.5 μl of 0.1 N NaOH. This solution wasincubated at room temperature for 1 h for CPT to be released from parentpolymer. 10 μl of 0.1 N HCl was then added to convert carboxylate CPTform to lactone form. 73.5 μl of methanol was subsequently added andmixture incubated for 3 h at room temperature. The sample was thencentrifuged and processed as above.

The average percentage injected dose (ID) of total CPT per gram oftumor, heart, liver, spleen, kidney and lung is shown in FIG. 19A. 4 hafter dosing, 5.3 and 5.2% ID of total CPT were present per gram oftumor in mice treated with MAP-CPT nanoparticles and targeted MAP-CPTnanoparticles, respectively. 24 h after treatment, 2.6% ID of total CPTremained per gram of tumor for mice treated with MAP-CPT nanoparticles,while 3.2% ID of total CPT were found per gram of tumor for micereceiving targeted MAP-CPT nanoparticles. These data show that targetednanoparticles of essentially the same size and surface charge of anuntargeted version do not increase tumor localization over that of anon-targeted version, and is consistent with observations reported byothers. Animals treated with MAP-CPT nanoparticles and targeted MAP-CPTnanoparticles gave similar distribution of CPT in heart, liver, kidneyand lung. There was, however, a comparatively significant amount oftotal CPT accumulation in the spleen for targeted MAP-CPT versusnon-targeted at 24 h. This effect has been observed previously forhumanized antibodies in mice.

FIG. 19B shows the average percentage of unconjugated CPT in each organfor tumor, heart, liver, spleen, kidney and lung. The percentageunconjugated CPT in heart, liver, spleen, kidney and lung were low atboth 4 h and 24 h, indicating fast clearance of free CPT from theseorgans. In tumor at 24 h, there were significantly higher percentages ofunconjugated CPT for both MAP-CPT and targeted MAP-CPT nanoparticlescompared to that at 4 h. The retention of free CPT within the tumorssuggests cellular accumulation of CPT. The targeted nanoparticles showslightly higher percentage of unconjugated CPT in tumors for mice overthe non-targeted nanoparticles at both 4 and 24 h.

The total concentration of CPT in plasma and percentage of total CPTthat is unconjugated in plasma were similar for both the MAP-CPT andtargeted MAP-CPT nanoparticles (FIG. 19C). After 4 h, 17 and 20 μg/ml oftotal CPT remained in plasma for MAP-CPT and targeted MAP-CPTnanoparticles respectively. After 24 h, the amounts of total CPTremaining in plasma were the same for both MAP-CPT and targeted MAP-CPTnanoparticles at 12 μg/ml. The percentage of total CPT that isunconjugated in plasma was below 3% for all conditions indicating smallrelease and fast clearance of unconjugated CPT from plasma (FIG. 19D).

Example 33 Treatment of Tumors with Targeted Nanoparticles

BT-474 tumor bearing NCr nude mice were treated with either MAP-CPT (5mg CPT/kg) or targeted MAP-CPT (5 mg CPT/kg, 29 mg Herceptin/kg). After4 and 24 h, mice were euthanized and tumors were removed and sectionedusing a cryostat to a thickness of 20-30 μm. The tumor sections wereplaced on Superfrost Plus slides (Fisher Scientific, Hampton, N.H.) andstored at −80° C. until time for processing. Slides were defrosted andtissue sections were fixed directly onto the slide for 15 min with a 10%formalin solution. The slides were then washed three times with PBS for5 min each, and blocked for 1 h in a 5% goat serum blocking buffer. CPTis naturally fluorescent with emission at 440 nm. To identify thelocation of MAP, the PEG within MAP was stained with a rat anti-PEGprimary antibody that recognizes internal PEG units at 14 μg/ml andincubated at 4° C. overnight. This was followed by three PBS washes and1 h incubation with 2 μg/ml of Alexa Fluor® 488 goat anti-rat IgMsecondary antibody. The slides were then washed three times with PBS,and incubated for 1 h with 2 μg/ml of Alexa Fluor® 633 goat anti-humanIgG secondary antibody to visualize Herceptin®. The slides were washedthree more times with PBS, mounted with a Prolong Gold Antifade reagentand stored at 4° C. until time for imaging. Images were acquired with aZeiss LSM 510 Meta Confocal Microscope (Carl Zeiss, Germany) using a 63×Plan-Neofluar oil objective. 2-Photon excitation at 720 nm (emissionfilter BP 390-465 nm) was used to detect CPT. Excitation at 488 nm(emission filter LP 530) and at 633 nm (emission filter BP 645-700 nm)were used to detect PEG and Herceptin respectively. All laser and gainsettings were set at the beginning of imaging and were unchanged. Imageanalysis was performed on Zeiss lsm image browser.

FIG. 20A shows tumor section of mice treated with MAP-CPT nanoparticlesafter 4 h. CPT signal was disperse. Signals for CPT and MAP appeared tocolocalize in the merged image. 24 h after injection, there wasaccumulation of CPT signal in the form of punctate spots (FIG. 20B). Themerged image suggests colocalization of CPT and MAP. For mice treatedwith targeted MAP-CPT nanoparticles, spots indicating CPT accumulationwere observed after both 4 and 24 h of treatment (FIGS. 20C and 20D). Inthe merged images, there was colocalization of CPT and MAP signals. Thepresence of Herceptin in targeted MAP-CPT nanoparticles is indicated bythe strong blue signals in the Herceptin channel compared with weakbackground signals in the non-targeted version.

Example 33 Antitumor Efficacy Study in Nude Mice

BT-474 is one of the most resistant breast cancers to anticancer drugsincluding camptothecin. To promote tumor growth in nude mice,17β-estradiol pellets were implanted into 7 weeks old NCr nude mice twodays prior to BT-474 tumor cell implantation. On day 0 (five days afterimplantation), tumor sizes of each group averaged 250 mm. Treatmentsbegan on day 1. Animals were randomly divided into nine groups with sixto eight mice per group and treated with either, MAP-CPT nanoparticlesat 1 mg or 8 mg/kg (CPT basis, in PBS, pH 7.4), CPT at 8 mg/kg(dissolved in 20% DMSO, 20% PEG 400, 30% ethanol and 30% 10 mM pH 3.5phosphoric acid), Irinotecan at 80 mg/kg (in 5 w/v % dextrose solution,D5W), Herceptin® at 2.9 or 5.9 mg/kg (in PBS, pH 7.4), targeted MAP-CPTnanoparticles at 0.5 mg CPT/kg and 2.9 mg Herceptin®/kg or 1 mg CPT/kgand 5.9 mg Herceptin®/kg (in PBS, pH 7.4), or saline. All treatmentswere freshly prepared and given via intravenous tail vein injection.Injections were standardized at 150 μl per 20 g body weight of mice.Treatments containing Herceptin® were given once per week for 2 weeks,all other groups were given once per week for 3 weeks. Tumor sizes wererecorded three times a week using caliper measurements (length×width²/2)and health of the animals was continuously monitored. Animals wereeuthanized when tumor volumes exceeded 1000 mm³. Six weeks afterbeginning of the treatment, animals were euthanized by CO₂ asphyxiation.The results from this study are presented in FIG. 21 and Table 7.

Tumors in the control group administered with saline grew rapidly (FIG.21). After 28 days, five out of eight mice had tumors exceeding sizelimit of 1000 mm³. All animals in this group were euthanized at thistime.

The group treated with CPT (8 mg/kg) resulted in no tumor inhibitioncompared to that of the control group (P>0.05). One and three treatmentrelated deaths were recorded on day 9 and 16, respectively, as well asfour euthanizations due to exceeding the tumor size limit on day 28.None of the animals survived to the end of the study.

Mice treated with Irinotecan at 80 mg/kg showed non-significant tumorinhibition compared to control group (P>0.05). One treatment relateddeath occurred on day 11. Tumor sizes reached an average of 575 mm³ bythe end of the study.

Animals receiving MAP-CPT nanoparticles at 8 mg CPT/kg showed highlysignificant tumor inhibition compared to that of control group (P<0.01).By the end of the study the mean tumor size reduced to 63 mm³ and threeout of the eight mice treated had tumor sizes regressed to zero.Although no deaths occurred in MTD study using non-tumor bearing NCrnude mice treated with MAP-CPT at 8 mg CPT/kg, one death occurred due toweight loss in this study on day 21. This may be because of the addedtumor burden in this study and/or because mice used in this study were 7weeks old, while in MTD studies, 12 weeks old mice were used.

Animals treated with Herceptin® at 5.9 mg/kg showed highly significanttumor inhibition compared to control group (P<0.01). On day 11 oftreatment, seven out of the eight animals treated had tumors regressedto zero. However, by the end of the study, two of the regressed tumorsrelapsed resulting in a total of five animals with tumors at zero volumeand a group mean tumor volume of 60 mm³ (FIG. 22). Mice treated withMAP-CPT nanoparticles at 1 mg CPT/kg were terminated early due to noobserved antitumor effects. Nevertheless, when

Herceptin® at 5.9 mg/kg was added as a targeting agent via a nitroPBA-BAlinker onto MAP-CPT nanoparticles at a low CPT dosage of 1 mg/kg(targeted MAP-CPT nanoparticles at 5.9 mg Herceptin®/kg and 1 mgCPT/kg), all tumors regressed to zero on day 9 of treatment and remainedat zero by the end of the study (FIG. 22). One non-treatment relateddeath occurred on day 37. This result is highly significant compared tocontrol group (P<0.01). The combination of results observed for thesethree groups indicate that there is improved efficacy of MAP-CPTnanoparticles by the addition of the Herceptin® targeting agent inaddition to the intrinsic activity of the Herceptin®.

Animals treated with Herceptin® at 2.9 mg/kg resulted in two animalshaving tumors regressed to zero by the end of study. However, theaverage tumor size increased from the beginning of the study to 278 mm³.This result is significant compared to control group (0.01<P≦0.05). Whenanimals were treated with targeted MAP-CPT nanoparticles containing0.5≦mg CPT/kg and 2.9 mg Herceptin®/kg, two tumors regressed to zero.The average tumor size reduced to 141 mm³ by the end of the study. Thisresult is highly significant compared to control group (P<0.01). Theseresults further suggest the benefits of targeting in tumor inhibition.

Five weeks after 17β-estradiol pellets implantation, several miceirrespective of treatment groups were observed to have distended bladderand abdominal bloating. This was likely due to the use of 17β-estradiolpellets, which causes hydronephrosis and urine retention in athymic nudemice. Six weeks after treatment, conditions became worse and more micewere observed to develop distended bladders and abdominal bloating, thusanimals were euthanized and the experiment was terminated.

TABLE 7 Anti-tumor efficacy study in NCr nude mice bearing BT-474xenograft tumors N_(TRD)/N_(NTRD)/ Mean tumor Median tumorN_(begin)/N_(end) ^(a) N_(euthan) ^(b) volume (mm³) volume (mm³)N_(reg to zero) ^(c) P vs saline^(d) MAP-CPT (8 mg/kg) 8/7 1/0/0 63 68 30.002 Irinotecan (80 mg/kg) 8/7 1/0/0 575 479 0 0.242 CPT (8 mg/kg) 8/04/0/4 808 417 0 0.781 Herceptin (5.9 mg/kg) 8/8 0/0/0 60 0 5 0.003Targeted MAP-CPT (1 mg CPT/kg, 8/7 0/1/0 0 0 7 0.001 5.9 mgHerceptin/kg) Herceptin (2.9 mg/kg) 6/6 0/0/0 278 245 2 0.026 TargetedMAP-CPT (0.5 mg CPT/kg, 6/6 0/0/0 141 187 2 0.005 2.9 mg Herceptin/kg)Saline 8/0 0/0/8 911 1087 0 — ^(a)N_(begin) is number of animals atbeginning of study, N_(end) is number of animals surviving to end ofstudy. ^(b)N_(TRD) is number of treatment related death, N_(NTRD) isnumber of non-treatment related death, N_(euthan) is number of animalseuthanized due to exceeding tumor size limit of 1000 mm³.^(c)N_(reg to zero) is number of animals with tumors regressed to zeroat the end of study.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the particles, compositions, systems andmethods of the disclosure, and are not intended to limit the scope ofwhat the inventors regard as their disclosure. Modifications of theabove-described modes for carrying out the disclosure that are obviousto persons of skill in the art are intended to be within the scope ofthe following claims. All patents and publications mentioned in thespecification are indicative of the levels of skill of those skilled inthe art to which the disclosure pertains. All references cited in thisdisclosure are incorporated by reference to the same extent as if eachreference had been incorporated by reference in its entiretyindividually.

It is to be understood that the disclosures are not limited toparticular compositions or biological systems, which can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. As used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. The term “plurality”includes two or more referents unless the content clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the disclosure pertains.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the specificexamples of appropriate materials and methods are described herein.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

What is claimed:
 1. A targeted nanoparticle comprising a mucicacid-containing polymer, a therapeutic agent, and a polymer containing anitrophenylboronic acid, said nitrophenylboronic acid being coupled tothe mucic acid-containing polymer with a reversible covalent linkage,said targeted nanoparticle being configured to present the polymercontaining the nitrophenylboronic acid to an environment external to thetargeted nanoparticle, wherein the polymer containing thenitrophenylboronic acid is conjugated to a targeting ligand at itsterminal end opposite the targeted nanoparticle, wherein the targetednanoparticle has one molecule of targeting ligand per targetingnanoparticle.
 2. The targeted nanoparticle of claim 1 wherein the mucicacid-containing polymer contains structural units comprising


3. The targeted nanoparticle of claim 1 wherein the polymer containing anitrophenylboronic acid is

wherein Functional group 2 comprises —B(OH)₂, —OCH₃, —OH, —COOH,aldehyde, amine, or azide, and wherein s=20-300.
 4. The targetednanoparticle of claim 1 wherein the targeting ligand is an antibody,transferrin, a ligand for a cellular receptor, a cellular receptorprotein, an aptamer, or a fragment of an antibody, transferrin, a ligandfor a cellular receptor, or a cellular receptor protein.
 5. The targetednanoparticle of claim 1 wherein the therapeutic agent is a smallmolecule chemotherapeutic agent or a polynucleotide.
 6. The targetednanoparticle of claim 5, wherein the polynucleotide is interfering RNA.7. The targeted nanoparticle of claim 5, wherein the small moleculechemotherapeutic agent is selected from camptothecin, an epothilone, ataxane and a polynucleotide or any combination thereof.
 8. The targetednanoparticle of claim 2 wherein the therapeutic agent is any one ofcamptothecin, an epothilone, a taxane, a polynucleotide or anycombination thereof; the polymer containing nitrophenylboronic acid is

wherein Functional group 2 comprises —B(OH)₂, —OCH₃, —OH, —COOH,aldehyde, amine, or azide, and wherein s=20-300, and the targetingligand comprises any one of an antibody, transferrin, a ligand for acellular receptor, or a cellular receptor protein, an aptamer, or afragment of an antibody, transferrin, a ligand for a cellular receptor,or a cellular receptor protein.
 9. The targeted nanoparticle of claim 2wherein the therapeutic agent is camptothecin, the polymer containingnitrophenylboronic acid is

wherein Functional group 2 comprises —B(OH)₂, —OCH₃, —OH, —COOH,aldehyde, amine, or azide, and wherein s=20-300, and the targetingligand comprises an antibody.
 10. A method of producing a targetednanoparticle comprising conjugating a nanoparticle comprising a polymercontaining a polyol with a polymer containing a nitrophenylboronic acidand conjugating the polymer containing the nitrophenylboronic acid to atargeting ligand, wherein the resulting targeted nanoparticle has nomore than one molecule of targeting ligand per targeting nanoparticle.11. The method of claim 10, wherein a. the polymer containing a polyolhas contains structural units comprising

b. the polymer containing a nitrophenylboronic acid is

wherein Functional group 2 comprises —B(OH)₂, —OCH₃, —OH, —COOH,aldehyde, amine, or azide, and c. the targeting ligand is an antibody,transferrin, a ligand for a cellular receptor, or a cellular receptorprotein, an aptamer, or a fragment of an antibody, transferrin, a ligandfor a cellular receptor, or a cellular receptor protein.
 12. The methodof claim 10, wherein the targeted nanoparticle further comprises a smallmolecule chemotherapeutic agent selected from camptothecin, anepothilone, a taxane and a polynucleotide or any combination thereof.13. A kit for producing a targeted nanoparticle comprising a targetednanoparticle comprising a polymer containing a polyol, a polymercontaining a nitrophenylboronic acid, a targeting ligand, andinstructions for producing a targeted nanoparticle having no more thanone molecule of targeting ligand per targeted nanoparticle.
 14. The kitof claim 13 wherein, a. the polymer containing a polyol comprising astructural of:

b. the polymer containing a nitrophenylboronic acid is

wherein Functional group 2 comprises —B(OH)₂, —OCH₃, —OH, —COOH,aldehyde, amine, or azide, and c. the targeting ligand is an antibody,transferrin, a ligand for a cellular receptor, or a cellular receptorprotein, an aptamer, or a fragment of an antibody, transferrin, a ligandfor a cellular receptor, or a cellular receptor protein.
 15. The kit ofclaim 13, further comprising a small molecule chemotherapeutic agentselected from camptothecin, an epothilone, a taxane and a polynucleotideor any combination thereof.
 16. A composition comprising the targetednanoparticle of claim 1 and a suitable vehicle and/or excipient.
 17. Thecomposition of claim 16, wherein the composition is a pharmaceuticalcomposition and the suitable vehicle and/or excipient is apharmaceutically acceptable vehicle and/or excipient.
 18. Thecomposition of claim 17, wherein the targeted nanoparticle furthercomprises ¹⁰B as part of at least one polymer containing anitrophenylboronic acid the composition being formulated for in vivoboron neutron activation therapy.
 19. A method to deliver a therapeuticagent to a target, the method comprising contacting the target with thetargeted nanoparticle of claim
 1. 20. The method of claim 19, whereinthe target is a cancer cell within the body of a mammal.
 21. A system todeliver a therapeutic agent to a target, the system comprising at leastone polymer containing a polyol and at least one polymer containing anitrophenylboronic acid capable of reciprocal binding through areversible covalent linkage, the at least one polymer containing apolyol and the at least one polymer containing a nitrophenylboronic acidto be assembled with the therapeutic agent in the targeted nanoparticleof claim 1 to be used to deliver the therapeutic agent to the target.22. A method to administer a therapeutic agent to an individual, themethod comprising administering to the individual the targetingnanoparticle of claim 1 comprising the therapeutic agent.
 23. A systemfor administering a therapeutic agent to an individual, the systemcomprising at least one polymer containing a polyol and at least onepolymer containing a nitrophenylboronic acid capable of reciprocalbinding through a reversible covalent linkage, the at least one polymercontaining a polyol and polymer containing a nitrophenylboronic acid tobe assembled with the therapeutic agent in the targeted nanoparticleaccording to claim 1, to be administered to the individual.
 24. Apopulation of targeted nanoparticles, each targeted nanoparticlecomprising a mucic acid-containing polymer, a therapeutic agent, apolymer containing a nitrophenylboronic acid, said nitrophenylboronicacid being coupled to the mucic acid-containing polymer with areversible covalent linkage, said targeted nanoparticle being configuredto present the polymer containing the nitrophenylboronic acid to anenvironment external to the targeted nanoparticle, wherein the polymercontaining the nitrophenylboronic acid is conjugated to a targetingligand at its terminal end opposite the targeted nanoparticle, whereinthe targeted nanoparticles and targeting ligands are in a ratio of 1 to1 in the population.